hydrogen exchange mass spectrometry of related proteins … · 2017-08-28 · although much ofthe...

B American Society for Mass Spectrometry, 2016DOI: 10.1007/s13361-016-1365-5

J. Am. Soc. Mass Spectrom. (2016) 27:1048Y1061

FOCUS: MASS SPECTROMETRY AS A PROBE OFHIGHER ORDER PROTEIN STRUCTURE: RESEARCH ARTICLE

Hydrogen Exchange Mass Spectrometry of Related Proteinswith Divergent Sequences: A Comparative Study of HIV-1 NefAllelic Variants

Thomas E. Wales,1 Jerrod A. Poe,2 Lori Emert-Sedlak,2 Christopher R. Morgan,1,3

Thomas E. Smithgall,2 John R. Engen1

1Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA2Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA3Present Address: Genzyme Corporation, Framingham, MA 01701-9322, USA

Abstract. Hydrogen exchange mass spectrometry can be used to compare theconformation and dynamics of proteins that are similar in tertiary structure. If relativedeuterium levels are measured, differences in sequence, deuterium forward- andback-exchange, peptide retention time, and protease digestion patterns all compli-cate the data analysis. We illustrate what can be learned from such data sets byanalyzing five variants (Consensus G2E, SF2, NL4-3, ELI, and LTNP4) of the HIV-1Nef protein, both alone andwhenbound to the humanHckSH3 domain. Regionswithsimilar sequence could be compared between variants. Although much of the hydro-gen exchange features were preserved across the five proteins, the kinetics of Nefbinding to Hck SH3 were not the same. These observations may be related to

biological function, particularly for ELI Nef where we also observed an impaired ability to downregulate CD4surface presentation. The data illustrate some of the caveats that must be considered for comparison experi-ments and provide a framework for investigations of other protein relatives, families, and superfamilies with HXMS.Keywords: Hck SH3, Protein conformation, Sequence conservation

Received: 4 December 2015/Revised: 11 February 2016/Accepted: 12 February 2016/Published Online: 31 March 2016

Introduction

Vish Katta and Brian Chait published [1] the first descrip-tions of direct detection of hydrogen/deuterium exchange

(HX) in proteins by mass spectrometry (MS). The 1991 Kattaand Chait paper is required reading in the HX MS field andwas, in many ways, ahead of its time. In the ensuing 25 years,many types of proteins have been analyzed with HX MS,ranging from small and simple proteins (e.g., insulin) all the

way to large, multi-protein complexes. Technology derived fromBrian Chait’s first studies has made complex analyses faster,more reliable, and robust, and has enabled comparative analysisof related proteins. Our laboratory has been interested in usingHXMS to compare proteins that are related in tertiary structure,but not necessarily in primary structure (e.g., [2, 3]). In under-taking these types of homologous protein comparison studies,we have come to realize just how complex this exercise can be.Studies of homologous proteins come with a set of distinctcaveats not present in other types of HXMS studies. As a tributeto the groundbreaking work of Brian Chait in the field of proteinstructure analysis by MS that was recognized with the 2015ASMS Award for a Distinguished Contribution in Mass Spec-trometry, we present a description of the caveats in homologous

Electronic supplementary material The online version of this article (doi:10.1007/s13361-016-1365-5) contains supplementary material, which is availableto authorized users.

Correspondence to: John R. Engen; e-mail: [email protected]

http://crossmark.crossref.org/dialog/?doi=10.1007/s13361-016-1365-5&domain=pdf

http://dx.doi.org/10.1007/s13361-016-1365-5

http://dx.doi.org/10.1007/s13361-016-1365-5

protein comparison experiments through our HXMS analyses offive closely related forms of the HIV-1 Nef protein.

Nef is a unique primate lentiviral accessory protein expressed inhigh concentrations shortly after infection and is essential for highviral loads in vivo [4–8]. Strains ofHIV-1 isolated from individualswho remain AIDS-free for many years in the absence of antiretro-viral drug treatment (long-term non-progressors or LTNPs) oftendisplay alterations to the nef gene [9–14], thereby implicating Nefas a critical virulence factor for the development of AIDS [15, 16].Targeted expression of Nef in transgenic mice induces a severeAIDS-like syndrome, identifying this protein as a key determinantof HIV pathogenicity [17]. Nef has no known catalytic activity,and functions instead by interacting with a wide variety of host cellproteins to enhance viral infectivity and promote immune escape[4, 18, 19]. The ability of Nef to interact with diverse host-cellprotein partners relates to its conformational plasticity.

Initial high-resolution structural data for Nef were ob-tained using deletion variants [20–23], as full-length Nef isprone to aggregation in the absence of partner proteins.Residues 2-39 and 159-173 were removed for early solu-tion NMR analyses of Nef [20, 23], while deletion variantslacking residues 54-205 [21] or 58-206 [22] providedcrystals suitable for structural determination. In these ini-tial X-ray crystal structures, however, residues 54-69 and149-178 remained disordered. The N-terminal region ofHIV-1 Nef (residues 1-25 and 2-57) was also investigatedby NMR [24, 25] and found to be mostly disordered. Allthe known structural information for HIV-1 Nef was as-sembled into a useful model of the full-length protein byGeyer and Peterlin in 2001 [26]. In this model, the largeinternal loop (residues 157-174) was inserted with a prob-able conformation before energy minimization.

Structural studies of HIV-1 Nef have also shed light onthe mechanisms by which different regions and conforma-tions of Nef regulate interactions with host cell effectorproteins [20–22, 27, 28]. First generation solution andcrystal structures revealed how Nef binds to SH3 domainsand forms homodimers because these interactions requiredonly the structured Nef core and not the N-terminal anchorregion or internal flexible loop absent from these structures[20–22]. More recently, the crystal structure of aNef:MHC-I peptide fusion protein in complex with theμ1 subunit of the clathrin adaptor AP-1, an interactionessential for MHC-I downregulation and immune escape,revealed important details of the Nef N-terminal anchordomain conformation [27]. Along similar lines, the crystalstructure of Nef bound to the AP-2 α-σ2 subunithemicomplex, an interaction responsible for CD4 down-regulation, revealed a biologically important conformationfor the Nef internal loop for the first time [28]. Finally, arecent structure of Nef in complex with the SH3-SH2regulatory region of Hck revealed a dramatic reorganiza-tion of Nef dimer interface [29] relative to earlier structuresof Nef bound to the SH3 domain alone. Taken together,these studies support the idea that the conformational plas-ticity of Nef is essential to its diverse functions.

Analysis of nef sequences from long-term non-progressors (LTNPs) revealed many scattered amino acidchanges relative to alleles from laboratory and other viru-lent HIV-1 strains [26, 30]. The conformational environ-ment of these residues is unknown but many of the aminoacid changes unique to LTNP Nef proteins fall outside ofthe structured core (Figure 1). We therefore hypothesizedthat the solution conformations of Nef from LTNPs may bedifferent from those of laboratory strains that are common-ly investigated (e.g., SF2, NL4-3) and that these confor-mational differences may help to explain the impaired Neffunction associated with the LTNP phenotype.

To address these questions, we used HXMS to compare theconformation of full-length Nef proteins from multiple HIV-1strains, including an LTNP-derived Nef protein (LTNP4) pre-viously shown to be defective for Src-family kinase activation[31]. A particular advantage of using HX MS for these com-parisons is that full-length Nef proteins, which are not amena-ble to NMR or crystallization due to poor solubility, can bereadily probed by HX MS because of the low protein concen-tration required for analysis (<25 μM) [32]). We employed HXMS to compare the conformational properties of recombinantNef proteins derived from the HIV-1 strains SF2, ELI, NL4-3,Consensus, and LTNP4. All five Nef proteins share a highdegree of sequence homology, especially in the structured coreregion (Figure 1a). However, the sequences are differentenough to complicate HX MS analysis at the peptide level.For example, the five Nef proteins do not share identical pepsindigestion patterns, making the comparison of regional HX fromprotein to protein difficult. Despite these challenges, we showthat these five Nef proteins are indeed different with respect todeuterium incorporation and SH3 binding characteristics. Inthe regions that could be compared across the proteins, thereare significant differences in conformation for the portions ofNef that have been defined as being functionally relevant. Moregenerally, our approach to the HX MS analyses of these Nefvariants provides a framework for investigation of other proteinrelatives, families, and superfamilies.

Materials and MethodsProtein Expression and Purification

SF2, ELI, and LTNP4 Nef proteins were expressed in Sf-9insect cells and purified as described previously [33]. Briefly, ahexahistidine tag was added to the N-terminus by PCR and thecDNA subcloned into the baculovirus transfer vector,pVL1392. A recombinant baculovirus was prepared bytransfecting Sf-9 insect cells with the transfer vector andBaculogold DNA according to the manufacturer’s protocol(BD-Pharmigen, San Diego, CA, USA). Recombinant Nefproteins were purified using immobilized metal affinity chro-matography. Purity and concentration were confirmed by SDS-PAGE, densitometry, Bradford assay, and electrospray massspectrometry. Consensus G2E and NL4-3 Nefs were expressedin E. coli and purified according to the protocol described in

T.E. Wales et al.: HX MS of Proteins with Different Sequence 1049

[31]. Human Hck SH3 domain was prepared by over expres-sion in E. coli [strain BL21 (DE3) pLysS] as described in [34].

Deuterium Exchange Reactions

Nef proteins in equilibration buffer (20 mM Tris HCl pH 8.3,100 mM NaCl, 3 mM DTT) were removed from –80 °C

storage and thawed on ice for approximately 10 min. The Nefproteins were then used as is or combined with the Hck SH3domain to investigate exchange in the complex.

Deuterium labeling for the peptide analysis of the fiveNef proteins alone was initiated with a 15-fold dilution of1 μL Nef (50 pmol) into D2O labeling buffer. Labelingproceeded for specific amounts of time (10 s to 4 h) and

(c)

(a)

Cons G2E SF2 ELI NL4-3 LTNP4

Cons G2E -- 89.5 79.5 88.3 88.0

SF2 89.5 -- 79.9 85.7 84.3

ELI 79.5 79.9 -- 79.0 78.0

NL4-3 88.3 85.7 79.0 -- 85.0

LTNP4 88.8 84.3 85.0 85.0 --

(b)

(d)

45

50

55

60

65

70

0.1 1 10 100 1000

Rel

ativ

e P

erce

nt D

eute

rium

Lev

el

Time (min)

ConsG2ESF2ELINL4-3LTNP4

NMR X-ray crystallography PxxP -helix -sheet

Cons MEGKWSKRSVSGWPAVRERMR----RAEPAAEGVGAVSRDLEKHGAITSSNTAATNAACAWLEAQEEE-E SF2 MGGKWSKRSMGGWSAIRERMRRAEPRAEPAADGVGAVSRDLEKHGAITSSNTAATNADCAWLEAQEEE-E ELI MGGKWSKSSIVGWPAIRERIR----RTNPAADGVGAVSRDLEKHGAITSSNTASTNADCAWLEAQEESDE NL4-3 MGGKWSKSSVIGWPAVRERMR----RAEPAADGVGAVSRDLEKHGAITSSNTAANNAACAWLEAQEEE-E LTNP4 MGGKWSKRSGVGWPRVRERMH----RAEPAADGVGAASRDLEKYGAITS-NTAANNADCAWLEAQEGEEE

Cons VGFPVRPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIYSQKRQDILDLWVYHTQGYFPDWQNYTPGPGIRY SF2 VGFPVRPQVPLRPMTYKAALDISHFLKEKGGLEGLIWSQRRQEILDLWIYHTQGYFPDWQNYTPGPGIRY ELI VGFPVRPQVPLRPMTYKEALDLSHFLKEKGGLEGLIWSKKRQEILDLWVYNTQGIFPDWQNYTPGPGIRY NL4-3 VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPGVRY LTNP4 VGFPVRPQVPLRPMTYKGALDLSHFLREKGGLEGLVYSQKRQDILDLWVYHTQGYFPDWQNYTPGPGIRY

Cons PLTFGWCFKLVPVEPEKVEEANEGENNCLLHPMSQHGMDDPEKEVLVWKFDSKLAFHHMARELHPEYYKDC SF2 PLTFGWCFKLVPVEPEKVEEANEGENNSLLHPMSLHGMEDAEKEVLVWRFDSKLAFHHMARELHPEYYKDC ELI PLTFGWCYELVPVDPQEVEEDTEGETNSLLHPICQHGMEDPERQVLKWRFNSRLAFEHKAREMHPEFYK-- NL4-3 PLTFGWCYKLVPVEPDKVEEANKGENTSLLHPVSLHGMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNC LTNP4 PLTFGWCFKLVPVEPEKVEEINEGENNCLLHPISLHGMDDPEREVLVWKFDSRLALHHMARELHPEYYKNC

M10G

G11V

S14P

A15R

I16V

R21HV37A

H44Y

S50N

T55N

E67G

A87GI91L

K96R

I105VW106Y

R109KE112D

I118V

A160I

S167CM172I

E178D

A180P

K182R

K188R

K192RF195L

D209N

SF2→LTNP4

Figure 1. Properties of the Nef alleles in this study. (a) Sequence alignment of Consensus (Cons) SF2, ELI, NL4-3, and LTNP4alleles of HIV Nef. Residues that differ in this alignment are highlighted with yellow. Elements of secondary structure are indicated onthe top of the alignment: green lines for α-helices, and blue lines for β-sheets. The PxxPmotif is indicated with a red line. Regions ofthe Nef sequence that have been studied using NMR [20, 23, 24] or X-ray crystallography [21, 22, 27, 28] are indicated under thesequence. (b) Sequence conservation between each of the Nefs, as calculated by ClustalW. (c) Tertiary locations of amino aciddifferences between SF2 and LTNP4 Nef, as an example comparison. Comparison was made on the Nef structural model by Geyerand Peterlin [26]. The amino acid changes are indicated near each colored ball, with red balls indicating dramatic substitutions andgrey balls indicating conservative substitutions. The structural core [22] has been indicated with a molecular surface. (d) Intactprotein HX MS of the five Nef proteins. Error bars are not indicated in this figure (see BMaterials and Methods^)

1050 T.E. Wales et al.: HX MS of Proteins with Different Sequence

the reaction was quenched with the addition of an equalvolume of quench buffer (150 mM potassium phosphate,pH 2.6). Samples were analyzed immediately following thequench step.

Protein:ligand HX MS experiments were performed atthe global level for Consensus G2E or NL4-3 Nef (asligand) bound to the human Hck SH3 domain (SH3:Nef),or at the peptide level for the human Hck SH3 domain (asligand) bound to Consensus G2E, SF2, NL4-3, or LTNP4(Nef:SH3). The SH3:Nef binding reaction was initiatedwith the mutual dilution of a stock solution of SH3 do-main (120 μM) with Consensus G2E (280 μM, using KD

of 0.85 μM [35]) or NL4-3 (48 μM, using KD of 1.4 μM[36]) in equilibration buffer. After preparation of thebinding reaction, the protein mixtures were equilibratedtogether at 4 °C for 1 h before the initiation of thelabeling reactions. The Nef:SH3 binding reaction wasinitiated with the mutual dilution of a stock solutions ofSH3 domain (180 μM with Consensus and SF2 or500 μM with NL4-3 and LTNP4) with Consensus NefG2E (50 μM), SF2 Nef (50 μM, using KD of 0.64 μM[35]), NL4-3 Nef (40 μM), or LTNP4 Nef (40 μM, usingKD of 0.25 μM [35]) in equilibration buffer. After prep-aration of the binding reactions, the protein mixtures wereequilibrated together on ice for 30 min before the initia-tion of the labeling reactions.

Deuterium labeling for global analysis of the SH3:Nef bind-ing reaction was initiated with a 15- fold dilution of 5 μL (60pmol Hck SH3) of the binding reaction into D2O labelingbuffer (20 mM Tris pD 8.3, 100 mM NaCl, 3 mM DTT).Under labeling conditions and assuming the KD values above0.85 μM and 1.4 μM for Consensus G2E and NL4-3 bound toHck SH3, respectively, Hck SH3was 82% bound to ConsensusNef and 60% to NL4-3 (calculations performed as described in[37, 38]). Labeling proceeded for specific amounts of time(10 s to 6 h) and then the reaction was quenched with theaddition of an equal volume of quench buffer (250 mM potas-sium phosphate pH 2.6). Samples were immediately frozen ondry ice and stored at –80 °C until mass spectral analysis.

Deuterium labeling for local analysis of the Nef:SH3binding reaction was initiated with a 15-fold dilution of2 μL of the Nef:SH3 binding equilibration mixture intoD2O labeling buffer. Under labeling conditions and assum-ing the listed KD values, Nefs were bound to the SH3domain at 84%, 87%, 91%, and 98% for Consensus, SF2,NL4-3, and LTNP4, respectively. Labeling proceeded forspecific amounts of time (10 s to 4 h) and the labelingreaction was quenched with the addition of an equal vol-ume of quench buffer (150 mM potassium phosphatepH 2.6). Samples were analyzed immediately followingthe quench step.

Analysis of Deuterium Incorporation

Global Deuterium Analysis Labeled proteins were injectedonto a self-packed POROS 20 R2 (Applied Biosystems,

Framingham, MA, USA) protein trap and desalted with0.5 mL H2O (0.05% TFA) at a flow rate of 500 μL/min. Theproteins were eluted into the mass spectrometer using a linear15%–75% acetonitrile gradient in 4 min at 50 μL/min using aShimadzu (Nakagyo-ku, Kyoto, Japan) HPLC (LC-10ADvp).The HPLC was performed using protiated solvents, whichresults in the removal of deuterium from the side chains andthe amino/carboxy termini that exchange faster than backboneamide hydrogens [39]. All steps were performed with theprotein trap, injector, and associated tubing submerged in anice bath. Mass spectral analyses were carried out with a Waters(Milford, MA, USA) LCT-PremierXEmass spectrometer with astandard electrospray source. The deuterium levels were notcorrected for back-exchange [39] and are, therefore, reported asrelative [32]. The average amount of back-exchange using theexperimental setup described above was 12%–15% based onanalysis of highly deuterated protein standards. The relativedeuterium incorporation for the SH3 domain was determinedby subtracting the mass of the unlabeled protein from the massat each labeling time. Each entire labeling time course exper-iment was performed in duplicate. After the elution of eachsample from the LC system into the mass spectrometer, aninfusion of myoglobin (500 fmol/μL) was used as a quasi-internal standard for calibration purposes. The error of deter-mining the deuterium level at any time point averaged ±0.4 Da.The isotope envelopes in bimodal patterns were fit with twoGaussian functions, the widths of which were estimated fromthe width of a single binomial isotopic envelope before andafter the appearance of the bimodal pattern. The unfolding ratefor SH3 domain spectra that presented evidence of a bimodalisotopic envelope was determined from the slope of pseudo-first-order kinetic plots of the decrease in the relative intensity of thelower mass envelope with time [34, 40, 41]. Slowdown factorswere calculated as described in [34] (the unfolding t1/2 for theSH3:Nef reaction divided by the t1/2 for the SH3 domain alone).

Local Deuterium Analysis Labeled samples prepared forthe analysis of deuterium peptic peptides were analyzedusing a custom Waters nanoACQUITY system [42]. La-beled samples were injected at a flow rate of 100 μL/mininto a 2.1 mm × 50 mm stainless steel column that waspacked with pepsin immobilized on POROS-20AL beads(prepared as described in [43, 44]) held at 15 °C. Underthese conditions, the digestion time was approximately 30 s.Peptic peptides were trapped and desalted on a VanGuardPre-Column trap (2.1 mm × 5 mm, ACQUITY UPLC BEHC18, 1.7 μm) for 3 min. The trap was placed in-line with aAcquity UPLC BEH C18 1.7 μm 1.0 × 100 mm column(Waters Corp.) and peptides were eluted into the massspectrometer with a 8%–40% gradient of acetonitrile over6 min at a flow rate of 40 μL/min. All mobile phasescontained 0.1% formic acid and the temperature of allcomponents, excluding the pepsin column, was 0.1 °C.Mass spectral analyses were carried out on a Waters QToFPremier. Peptides produced from online pepsin digestion


were identified using Waters ProteinLynx Global Serverver. 2.4 with Identity Informatics software. Deuterationlevels were calculated using Waters DynamX software bysubtracting the centroid of the isotopic distribution for pep-tide ions of an undeuterated sample from the centroid of theisotopic distribution of peptide ions from a labeled sample.The deuterium levels were not corrected for back-exchange[39] and are, therefore, reported as relative [32]. Each entirelabeling time course experiment was performed in dupli-cate, and the deuterium uptake values in uptake curves (asin Supplemental Figure S4) represent the average of theduplicate measurements.

Results and DiscussionThe Proteins That Were Compared

HX MS has been used previously to study HIV-1 Nef.The conformation of both full-length HIV-1 Nef (strainSF2) and SIV Nef (mac239) were first studied in solution[45] and found to be consistent with the Geyer-Peterlinmodel of full-length Nef [26]. One significant advantageof HX MS is that the experiments are performed at proteinconcentrations of 25 μM or less [45] where full-lengthNef proteins are not prone to aggregation. Therefore, thedynamics of entire full-length protein—as opposed todeletion variants or shorter, more soluble constructs re-quired for NMR—could be analyzed. Successful bindingexperiments utilizing HX MS include analysis of SF2 Nef[31] and Consensus G2E Nef binding to various Hck SH3domains [46]. All prior Nef:SH3 binding HX MS exper-iments were done at the intact protein level.

Previous comparison of HIV-1 Nef alleles from eightLTNPs identified a unique Nef protein (LTNP4) withimpaired function in terms of Src-family kinase activationand association with the Hck SH3 domain [31]. These resultsled us to question the mechanism behind the defect in NefLTNP4 function despite sequence similarity with the otherpatient and lab-adapted alleles. Amino acid sequence align-ments of four well known laboratory strains of Nef withLTNP4 showed scattered differences across the backbone, withthe sequence of ELI the most unique of the five proteins(Figure 1a). This observation is consistent with the fact thatELI Nef is derived from the HIV-1 D subtype, while the otherlab alleles are B clade. Calculations of sequence conservation(Figure 1b) yielded sequence identities that ranged from 79.0%to 89.5%, with the lowest identity between NL4-3 and ELI andthe highest between SF2 and Consensus G2E. The location ofsequence differences do not map to obvious motifs involved inknown Nef partner protein interactions, and thus did not ex-plain the observed differences in SH3 domain binding orfunction [31]. Figure 1c shows an example for the amino aciddifferences between SF2 and LTNP4 Nef represented as redand grey balls on the Nef model structure. Many of the aminoacid changes are conservative (e.g., Lys to Arg, Asp to Asn, Ileto Leu, grey balls) and not predicted to affect the overall

conformation. The significant differences in sequence (e.g.,Trp to Tyr, or Glu to Gly, red balls), which may change theconformation and therefore the function/binding were general-ly outside of the stable structured core of Nef and not inlocations predicted to impact function.

HX MS on the Intact Protein Level

We hypothesized that higher-order protein conformationmay play a significant role in the function of Nef. This ideais based on the observation that seemingly subtle differ-ences in the primary structure for the five forms of Nefdid not map to interaction surfaces and, therefore, mayallosterically influence tertiary structure and conformationaldynamics. Because the incorporation of deuterium into aprotein backbone in solution is a function of both solventaccessibility and hydrogen bonding, any change to either ofthese parameters alters the level of deuteration [47]. Ac-cordingly, HX MS was used in order to determine if subtleand scattered differences in primary structure translated intodifferences in tertiary structure and, therefore, HX.

Initially, we chose to investigate the relative deuteriumlabeling of all five Nef proteins at the intact protein level,similar to the famous Katta and Chait studies of ubiquitin [1],to provide evidence for global differences in tertiary structure.Immediately, we encountered problems in data interpretation.First, the maximum number of backbone amide positions thatcould be labeled in each Nef protein was different because ofdifferences in the length of the proteins and in sequence differ-ences that changed the number of proline residues [number ofbackbone amide hydrogens in each protein in parenthesis:Consensus-G2E (201), SF2 (213), ELI (207), NL4-3 (208),and LTNP4 (196)]. In an attempt to compensate for thesedifferences, we converted the relative deuterium incorporationto relative percent deuterium level to compare exchange intothe proteins at the global level (Figure 1d). This approachinitially suggested the presence of global differences in deute-rium incorporation. The largest difference in exchange (13%)was at the 1 min time point between SF2 and ELI Nef. Inter-estingly, LTNP4 had a similar deuterium incorporation profilecompared with the other three laboratory strains. Overall, ELINef—which does not bind to Hck SH3 because of a singlepoint mutation in the RT-loop binding pocket [48]—incorpo-rated less deuterium compared with the other four Nefs. How-ever, investigations of this nature, meaning HX MS studies ofintact proteins with variable sequence, are fraught with prob-lems. Differences in sequence change forward- and back-exchange rates, and as we describe below, interpretation ofthe data as presented in Figure 1d is generally so confoundedwith problems that it becomes meaningless.

Difficulties in Comparing HXMSWhen Sequence isVariable

The required considerations for correct interpretation ofHX MS data from proteins with different sequences apply


to both intact proteins and to the peptic peptides producedin many pepsin-level experiments. Because intact proteinHX MS lacks spatial resolution, many studies involvedigestion of deuterium-labeled proteins into peptides withthe acid protease pepsin and then MS analysis of thedeuterium level of each peptide as a function of time[39]. Major problems in data interpretation arise when thesequences of the proteins one wishes to compare are notidentical and no correction has been made for back-ex-change. To correct for back-exchange, a fully deuteratedreference protein must be analyzed (recently reviewed inreference [49]) and a correction equation [39] applied. Inthe case of Nef, fully deuterated reference proteins couldnot be prepared and, therefore, all deuterium levels arereported as relative [32]. As a result, data interpretation isconfounded by the following variables: (1) both forward-and back-exchange rates change with sequence, (2) extentand position of pepsin digestion can be different, (3) theLC retention time of peptides with different sequences canbe different and, therefore, the amount of time such pep-tides are exposed to quench conditions and undergo back-exchange can be variable, (4) direct comparison of deute-rium levels for a given region of a protein, often done forrelative deuteration comparisons, may not be possible ifthe identical peptides are not found. Point 1 applies to bothintact protein and peptide HX MS whereas points 2–4apply to peptide-level experiments. Of course, back-exchange correction would make the data analysis muchmore straightforward, as would single amino acid HXresolution; unfortunately, neither of these is possible formany proteins, including Nef. Our intent here is not toaddress how to analyze sequence variable proteoforms byapplying back-exchange corrections or by obtaining singleamino acid resolution but rather to use this example of Nefproteins to discuss what one can do with relative deuteriumincorporation data when sequences are variable. Detaileddescriptions of methods for preparing totally deuteratedcontrols and why those methods often fail for many pro-teins, as well as descriptions of the many issues related tothe inability to perform single amino acid resolution stud-ies in HX MS with ETD and related non-ergotic fragmen-tation process are topics for other future articles.

Hydrogen exchange rates for backbone amide hydrogensvary as a function of all levels of structure. Sequence affects theexchange rates because the amino acid sidechain on either sideof a backbone amide hydrogen exerts influence over the kinet-ics of the exchange reaction; the extent of this effect can becalculated for any sequence [50, 51]. Theoretical calculationsof sequence-dependent exchange rates do not account for theinfluence imparted by secondary, tertiary, and quaternarystructure. To determine how much influence sequence mayhave over the exchange of the five forms of Nef, the theoreticalbackbone amide hydrogen exchange rates for all five Nefproteins were calculated according to [50]. Again, these calcu-lations assume that the sequence adopts a completely

unstructured form devoid of secondary, tertiary, and quaternarystructure. Both back exchange at quench conditions [pH 2.5, 0°C – reported as back exchange half-life, or BE t1/2] andforward exchange at the experimental conditions [pD 8.3, 21°C – reported as kint] were calculated. The full results plotted asa function of amino acid sequence are shown in SupplementalFigure S1. The average (across all backbone amide hydrogens)BE t1/2 values for each protein were: Consensus-G2E:159.08 min; SF2: 159.55 min; ELI: 170.74 min; NL4-3:165.85 min; LTNP4: 164.63 min. The average forward-exchange (kint) for each of the Nef proteins were: Consensus-G2E: 2649.89min–1; SF2: 2786.23 min–1; ELI: 2858.25min–1;NL4-3: 2827.42 min–1; LTNP4: 2848.73 min–1. If we assumethat all of the Nef proteins become equally deuterated beforeexposure to back-exchange conditions, ELI Nef would retainthe most label followed by NL4-3, LTNP4, SF2, and Consen-sus G2E. Further, if we assume that secondary, tertiary, andquaternary structures play no role in the deuteration,Consensus-G2E and SF2 Nef would bemore slowly deuteratedthan the other three Nef proteins. In reality, of course, second-ary, tertiary, and quaternary structures strongly influence theexchange rates and rankings based solely on forward- andback-exchange are therefore not observed (Figure 1d). Thesecalculations are merely hypothetical examples to illustrate thepoint that with divergent sequences, one should not expect toobserve the same level of deuterium incorporation by MS.

Digestion into peptides further complicates comparison ofthe five Nefs. The peptides that were followed during theseexperiments (encompassing 98%, 88%, 86%, 90%, and 82% ofthe amino acid sequences for Consensus G2E, SF2, ELI, NL4-3, and LTNP4 Nefs, respectively, not considering purificationtags) are shown in the peptide maps in Supplemental Figure S2.This figure clearly shows that sequence variability gives rise tomarkedly different digestion patterns. Two specific examplesof the theoretical calculations just described for intact proteinsare shown for peptides in Figure 2; we will consider the effectsof back-exchange first. It is a safe, but not always correct (seebelow), assumption that after digestion, peptides have no sec-ondary, tertiary, or quaternary structure to influence exchange.If this is true, then back-exchange during analysis, therefore,should be entirely a function of sequence and environmentalconditions. The half-life for deuterium in backbone positions torevert to hydrogen for any of the Nef proteins spans threeorders of magnitude between 1 and 1000 min (SupplementalFigure S1A). No peptide in this experiment underwent greaterthan 12 min exposed to back-exchange conditions (i.e., beingin an all hydrogen environment from cold injection of thequenched reaction and online digestion, through UPLC sepa-ration, and ultimately MS detection). Therefore, we have con-servatively doubled this time and have used a BE t1/2 of 30 min(represented as a purple dashed line in Figure 2a and Supple-mental Figure S1A) as an artificial cutoff for amino acidresidues that could Bpose a problem.^ BPosing a problem^


10

100

1000

BE

t½

(min

)

Amino Acid Sequence Number

V G F P V R P Q V P L R P M TV G F P V R P Q V P L R P M TV G F P V R P Q V P L R P M TV G F P V T P Q V P L R P M TV G F P V R P Q V P L R P M T


0807

(a)

(b)

H D

H D

D H

Back e

xchange

Forw

ard e

xchange

100

1000

10000

k in

t(m

in-1

)

V G F P V R P Q V P L R P M TV G F P V R P Q V P L R P M TV G F P V R P Q V P L R P M TV G F P V T P Q V P L R P M TV G F P V R P Q V P L R P M T


0807


100

1000

10000

k in

t(m

in-1

)

K R S V S GK R F P G GK S S I V GK S S V I GK R S G V G

W P A V RW S A I RW P A I RW P A V RW P R V R


616


Figure 2. Theoretical amide hydrogen exchange rates for selected peptides/regions of all five Nef proteins. The completecalculations for the entire proteins are found in Supplemental Figure S1. Consensus G2E: solid orange circles; SF2: hollow greensquares; ELI: hollow blue triangles; NL4-3 grey x marks; LTNP4: hollow red circles. (a) The half-life of back-exchange (BE t1/2, inminutes) at pH2.5 and 0 °C in the PxxPxR region of Nef. The purple dashed linemarks BE t1/2 value of 30min. Proline residues, whichdo not have backbone amide hydrogens, are indicated at the top of the BE t1/2 axis but do not have a numeric value. (b) Rates offorward exchange (kint, in min–1) at pD 8.3 and 21.0 °C for peptides covering the PxxPxR region (top panel) and an N-terminal region(bottom panel) of Nef. Proline residues, which do not have backbone amide hydrogens, are indicated at the bottom of the kint axis butdo not have a numeric value


means that in the analysis, any backbone amide hydrogen witha value that is below the 30 min back-exchange line could loseenough label during analysis that one might expect to observechanges in the measured mass. An example of where thiscalculation could become important is a peptide that coversthe PxxPxR region of Nef. This region is essential for SH3domain binding [52] and is represented in all five Nef proteinsby a 15 amino acid peptide with identical termini. However,

only four out of the five proteins have identical sequences.NL4-3 has a threonine at position 6 instead of the conservedarginine. Arguably, this single difference is significant in thatwhat was once a large charged side chain has now beenreduced to a shorter polar side chain. Additionally, there is alsothe ability of the arginine ε−NH to retain deuterium label underquench conditions and in the timescale of the LC MS analysis.Figure 2a, a larger version of this region from Supplemental

5 se

c10

sec

1 m

in10

min

1 ho

ur4

hour

s

-S

H3

+ S

H3

No coverage

Gap in alignment

ConsG2E SF2 NL4-3 LTNP4 ELI

50

100

150

200

50

100

150

200

1

2

3

4

(a)

(b)

Relative P

ercent Deuterium

62.5

25.037.550.0

12.5

75.0100.0

1

2

3

4

Figure 3. Vertical heatmaps of the deuterium incorporation into peptides of HIV variants. The peptic peptides for Nef free in solution(a) and bound to the HckSH3 domain (b) are shown as colored bars extending from the N-terminus (top of each panel) to the C-terminus (bottom of each panel). The length of each peptide is proportional to the vertical height, and overlapping peptides areexcluded from this view for clarity. The location of every 50th amino acid is shown with horizontal lines. The deuterium level at eachexchange-time (from left to right: 5 s, 10 s, 1 min, 10 min, 1 h, and 4 h, as shown at top of ConsG2E panel) is shown in relativepercentage, according to the color code shown. Peptide regions 1–4 as discussed in the text are indicated as vertical bars to theright of the ConsG2E panels. More details about the peptides used to create this figure are shown in Supplemental Table S1.


Figure S1, shows the calculation. Even when the sequencechanges (R to T), altered back-exchange would not substan-tially change the relative deuterium incorporation graph. Back-exchange for the backbone amide hydrogens in this sequence(71VG..MT85, referred to hereafter as Peptide 1, see Supple-mental Figure S3) is slow and well above the dotted purple line.Therefore, we have chosen to designate this region as accept-able for comparison despite the sequence differences. The MSresults for deuteration of this peptide can likely be comparedand the minor difference observed in the uptake graph (Sup-plemental Figure S3, peptide 1) can be considered asmeaningful.

As illustrated with the preceding example, theoreticalcalculation of back-exchange becomes relevant when com-paring a specific peptide region that does not have thesame amino acid sequence or length. Back-exchange dur-ing analysis, in addition to being affected by sequence, canbe affected by the actual conditions of the analysis. Arecent study [53] illustrated this point with a series ofdeuterated peptides of varying lengths derived from asingle protein as a means to increase special resolution.Rates of back-exchange for the same sequence but ofvarying length can be different. Peptides of varying lengthsand hydrophobicity theoretically have differing retentiontimes during the LC separation step and subsequentlyvarying percentages of back-exchange based on the com-bined effects of the sequence differences and the amount oftime exposed to quench conditions. There may also besecondary structure even in quench conditions and se-quence may affect secondary structure and the theoreticalamount of back-exchange that is to be expected. Therefore,even peptides that are identical in much of the sequencebut differ by a few amino acids in length should be treatedas suspect. Supplemental Figure S3 shows various exam-ples of different scenarios in the comparison of peptidesacross all five of the Nef proteins. Peptides 3 and 4 inSupplemental Figure S3 differ by only a few amino acids,but would a comparison of relative percent deuteration bevalid given the difference in length? Another example is atthe N-terminus. A peptide region in the N-terminus (far leftof Supplemental Figure S3) certainly cannot be compareddue to changes in peptide length and sequence. For thisregion, the relative deuterium uptake graph and any figurederived from it (e.g., Supplemental Figure S3, top, andFigure 3) cannot be interpreted with certainty becausedifferences in deuterium labeling could be due to differ-ences in back-exchange rather than differences in confor-mation. As already mentioned above, correction for loss ofdeuterium during analysis (if possible) and not relyingmerely on measurements of the relative amount of deute-rium incorporation is one solution to such comparisondilemmas.

We also want to mention that the intrinsic rate of back-bone amide hydrogen forward-exchange during the label-ing step (kint) is another important consideration. The kint

is a fundamental property of the primary structure of aprotein and is independent of secondary, tertiary, and qua-ternary structures. Differences in the intrinsic rate can alsobe viewed as an initial level of comparison amongst ho-mologous proteins as the primary structure not only dic-tates kint, but it also drives/creates both secondary andtertiary structures. For HX MS investigations of proteinvariants measured under conditions of equivalent pH, ionicstrength, and temperature, would the kint for the backboneamide hydrogens within structurally homologous regionsalter what one would measure by MS for such a region?For the Nef variants, the intrinsic rates of exchange for allbackbone amides were calculated at experimental condi-tions of labeling (pD 8.3 and 21 °C), as shown in Supple-mental Figure S1B. As in back-exchange, those peptidesthat have similar sequences will have similar values forkint, as illustrated with the top panel of Figure 2b [note thatthe scenario in Figure 2b is what may happen in HX MS ofpoint mutants, again emphasizing that even small changesto sequence must be considered in data interpretation]. Forthose regions where there is more sequence variability,such as the N-terminus of these five Nef proteins, compar-ison of HX data in this region across all of the variants ismore difficult. Assuming no secondary, tertiary, or quater-nary structures, the calculations for a region from7KR..VR17 in Nef (Figure 2b, bottom panel) shows thatthe rate of exchange for a single backbone amide hydrogencan vary by two orders of magnitude. Imagine then ascenario in which the kint was different between two pro-tein variants in a structurally homologous region (e.g., asolvent-exposed loop). Differences in the amount of deu-terium incorporation could be driven solely by these se-quence differences in regions where secondary, tertiary,and quaternary structures do not override (by slowing)the rate of exchange calculated from sequence alone.

What Can be Compared?

Considering both the differences in the peptides producedduring online digestion and the differences in the rates ofintrinsic forward- and back-exchange over the five Nefsequences, we were only able to do head-to-head compar-isons for just under 30% of the Nef sequence across allfive proteins. Not surprisingly, this mostly included re-gions of the protein core where the sequence was lessvariable (see Figure 1c as an example). Given that somecomparisons of data are not valid, another complication ishow to faithfully indicate differences in deuterium incor-poration for the five proteins while visually representingthe deuterium incorporation data in the most effectivemanner for valid cross-variant comparison.

We have chosen to present the peptide data as relativepercent deuterated (calculated from the measured deuteri-um level divided by the deuterium level if all possiblebackbone amide hydrogen positions were deuterated).However, the comparison of deuterium exchange data


for peptides of varying lengths and sequences using arelative percent deuterated scale generally is not valid. Adetailed section at the beginning of the SupplementalMaterial has been included to illustrate pitfalls in compar-isons of relative percent deuterated data. We describe two

instances where the direct comparison of different pep-tides is not valid and a third that illustrates that thecomparison does have merit in certain instances (e.g., acomparison of percent deuterium incorporation can be avalid exercise for two peptides of identical sequence with

0

Time (min)

20

40

60

80

100

0.01 0.1 1 10 100 1000

SF2

- SH3

+ SH3

Rel

ativ

e P

erce

nt

Deu

teri

um

0

20

40

60

80

100

0.01 0.1 1 10 100 1000Time (min)

- SH3

+ SH3

LTNP4

0

Time (min)

20

40

60

80

100

0.01 0.1 1 10 100 1000

LTNP4

- SH3

+ SH3

0

20

40

60

80

100

0.01 0.1 1 10 100 1000

NL4-3

- SH3

+ SH3

0

20

40

60

80

100

0.01 0.1 1 10 100 1000

- SH3

+ SH3

Consensus G2E

VGFPVRPQVPLRPMTVGFPVRPQVPLRPMTVGFPVTPQVPLRPMTVGFPVRPQVPLRPMT

ConsG2ESF2

NL4-3LTNP4

0

20

40

60

80

100

0.01 0.1 1 10 100 1000

- SH3

+ SH3

Consensus G2E

0

20

40

60

80

100

0.01 0.1 1 10 100 1000Time (min)

Rel

ativ

e P

erce

nt

Deu

teri

um

- SH3

+ SH3

SF2

0

20

40

60

80

100

0.01 0.1 1 10 100 1000

- SH3

+ SH3

NL4-3

0

20

40

60

80

100

- SH3

+ SH3

NL4-3

(a)

(b)

.iii.i

.vi.ii

.iii.i

.vi.ii

LWVYHTQGYFWIYHTQGYFLWIYHTQGYFWVYHTQGYF

ConsG2ESF2

NL4-3LTNP4

v.

v.

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0

0.01 0.1 1 10 100 1000Time (min)

SF2ConsG2E

NL4-3LTNP4R

elat

ive

dif

fere

nce

(D

a)

-2.0

-1.5

-1.0

-0.5

0

Time (min)0.01 0.1 1 10 100 1000

SF2ConsG2E

NL4-3LTNP4R

elat

ive

dif

fere

nce

(D

a)

Figure 4. Regions of Nef that showed a difference in peptide HX upon Hck SH3 binding. (a) Deuterium incorporation plots for thepeptide spanning the PxxPxRmotif and identified as peptide 1 on Figure 3. The location of this peptide is colored red in the structuralmodel (PDB 1EFN) at top left above a sequence alignment. (b) Deuterium incorporation plots for the peptide identified as peptide 2 inFigure 3. The location of this peptide is colored blue in the structural model at top left above a sequence alignment. For both panels aand b, results are shown for Consensus G2E (subpanel i in both a and b), SF2 (ii), NL4-3 (iii), and LTNP4 (iv). The calculated relativedifferences (Hck SH3 bound Nef – Nef alone) for each of the Nefs are shown in subpanel v. Error bars represent the minimum andmaximum data achieved from duplicate measurements. All deuterium incorporation plots are in Supplemental Figure S4


slightly different lengths at either the N- or C-terminibecause the total possible exchange sites and amino acidlengths of the peptides are similar). In illustrating peptide-level data for the Nef proteins, we have chosen to employboth vertical heat maps (Figure 3) and the more traditionaldeu t e r i um inco rpo r a t i on p lo t s (F igu r e 4 , andSupplemental Figures S3 and S4).

It is possible and informative to compare changes to thesefive Nef proteins when interacting with a protein partner, theHck SH3 domain. Comparisons of the effects of binding arenot influenced by all of the caveats discussed above becausethe peptides in free and bound forms remain the same. Thepattern and extent of pepsin digestion is much less affected bythe presence of other proteins during digestion. Therefore,relative deuterium measurements in binding experiments of

various related proteins can provide reliable information abouthow sequence variation affects interactions even when directcomparisons of the exchange of the related proteins suffersfrom multiple data interpretation complications.

Comparison of Nef Proteins at the Peptide Level

The results of the Nef HX digestion experiments, both free andbound to the Hck SH3 domain, are visually summarized inFigures 3 and 4, and all deuterium incorporation plots can befound in Supplemental Figure S4A–E. Figure 3 makes it easyto quickly see how the Nef proteins become deuterated (panelA) and where there are/are not differences upon binding (whencomparing panel A and B, as described below). As discussedabove, a direct comparison across all peptides derived fromeach of the Nef proteins—that is comparing data between theproteins horizontally across Figure 3—is not a valid exercise.There are regions that meet the criteria outlined above for validcomparison of deuterium uptake, including the Nef core andfour peptide regions (labeled 1–4 in Figure 3 and in Supple-mental Figure S3)

Peptide regions 1, 2, and 3 have nearly the same deu-terium incorporation profiles across all five Nef proteins.Region 1 covers the PxxPxR motif responsible for bindingto Src-family kinase SH3 domains, and there were onlyslight differences in deuterium exchange for the free Nefproteins in this region. Regions 2 and 3 are both within thefolded core of each Nef variant, and the exchange datawere largely similar here; this is not unexpected as theseregions have nearly identical amino acid sequences. Therewas no sequence coverage for ELI Nef in the peptide 2region, so a complete comparison is not possible. Peptideregion 3, which encompasses β-sheets 1 and 2, showsnearly identical deuterium incorporation for the three B-clade laboratory strains, while both ELI (clade D) and theprimary LTNP4 Nef proteins are distinct. However, thisregion of the Nef core has not been ascribed any specificfunction.

Peptides in area 4 overlap almost completely with a shortNef α-helix (helix 4) that is induced when in complex with theAP-2 endocytic adaptor protein [28]. This interaction is

essential for Nef to remove CD4 receptors from the surface ofinfected cells through the AP-2 endosomal trafficking pathway.The location of the Nef area 4 peptide is modeled on the crystalstructure of the Nef:AP-2 complex in SupplementalFigure S5A. In this region, the rate of deuterium incorporationis indistinguishable for Consensus G2E, SF2, and LTNP4,whereas NL4-3 may be slightly reduced. However, deuterationof this region is significantly delayed for ELI in comparison tothe other forms, raising the question of whether this differenceis reflected in Nef function related to CD4 downregulation. Toaddress this possibility, we compared the extent of CD4 down-regulation by each of these Nef proteins in the SupT1 T-cellline by flow cytometry. As shown in Supplemental Figure S5B,ELI Nef was substantially impaired for CD4 downregulation,with only 14% of the cell population demonstrating reducedcell-surface CD4 levels compared with 54% to 86% for theother Nef variants tested. Differences in deuterium uptake inthis regionmay reflect altered dynamics that may in turn impactinteraction with AP-2 and subsequent CD4 downregulation.

Comparison of Nef Variants in the SH3-Bound State

As alluded to above, differences in HX observed upon partnerprotein binding should not be affected by alterations to intrinsicexchange rates as a result of sequence changes because data arecompared for the same protein in unbound versus bound states.To illustrate this point, we explored the HX MS changesresulting from SH3 domain binding to each of our Nef variants.Nef modulates signaling of pathways linked to Hck and otherSrc-family kinases by binding to their SH3 domains, leading toconstitutive kinase activation. A previous study has shown thatthe SF2, ELI, and LTNP4 variants of Nef have different bind-ing affinities for the SH3 domain of Hck [31]. In that study, thechange in deuteration of Hck SH3 when bound to a ligand wascompared with deuteration of unbound Hck SH3 to calculate aslowdown factor (see the BMaterials and Methods^ section). Alarger slowdown factor means tighter binding. SF2 Nef boundtightly to SH3 (large slowdown factor), LTNP4 Nef boundweakly, and ELI Nef did not bind at all, and these bindinginteractions correlated well with Hck activation by each ofthese Nef proteins [31]. In the current study, we included twoadditional forms of Nef: Consensus G2E and NL4-3, neither ofwhich had previously been studied by HX MS (SupplementalFigure S6). HX MS data suggest that Consensus-G2E Nefbinds as tightly to SH3 as SF2, whereas NL4-3 binds less welland in a manner that is similar to LTNP4 Nef. ELI Nef does notbind at all, consistent with previous work [48]. Although thebinding activities of these five Nef alleles towards the Hck SH3domain vary, HX data from the Nef SH3-binding region (pep-tide 1 in Supplemental Figure S3) are nearly identical, consis-tent with the high degree of sequence conservation in this area.Therefore, other factors besides the presence of the PxxPxRsequence must substantially contribute to the interaction be-tween Hck SH3 and Nef. Indeed, early structures of theNef:SH3 complex revealed a major role for the three-dimensional fold of the Nef in complex formation [21]. By


extension, intrinsic protein dynamics are also likely to have amajor impact, as well as other protein–protein interactionsessential to Nef function.

We next examined the impact of SH3 binding on each Nefprotein at the peptide level by digesting the deuterium labeledSH3-bound Nef proteins and comparing those data to theunliganded proteins. ELI Nef does not bind to Hck SH3 [31]and served as a negative control. In addition to looking forchanges in the PxxPxR region, we were interested to see ifthere were any other changes to Nef conformation in thepresence of the SH3 domain. Figure 3b shows the relativepercent deuterium for Nef peptides derived from the SH3-bound form, which can be compared directly to the relativepercent deuterium level of the peptides derived from the apoform in Figure 3a. In this Figure, comparisons of the relativedeuterium level in the same protein between panel A and B(vertical) is a valid comparison, whereas comparison acrossalleles (horizontal) is subject to the caveats described above.That said, visual inspection reveals that deuterium uptakeacross most of the Nef proteins remains unchanged as a func-tion of SH3 binding. However, two regions show significantdifferences across the Nef proteins, and these are described indetail in the next section.

Two peptide regions with similar sequences could be reli-ably compared across the Nef proteins as a function of SH3binding (peptides 1 and 2; Figures 3 and 4). Peptide 1, whichencompasses the PxxPxR motif, showed an initial decrease indeuterium incorporation in the presence of Hck SH3 across allfour Nef proteins (Figure 4a). The initial protection from ex-change at the early time points is evident for all of the Nefproteins tested (compare gray lines to colored lines). Interest-ingly, there is also a difference in the timing of the dynamicbehavior of the backbone in this region for each Nef variant inthe presence of the SH3 domain. This is apparent when thedifferences between the deuterium incorporation curves (SH3-bound minus free) are plotted on the same axes (Figure 4a,panel v). LTNP4 becomes labeled more quickly relative to SF2and NL4-3, whereas Consensus G2E is intermediate. Peptide 2,which encompasses the C-terminal half of Nef α-helix 2 andfaces the Nef:SH3 interface, exhibits a decrease in deuteriumincorporation for the longer exchange time points. The differ-ence between Nef bound and free for this peptide (Figure 4b,panel v) indicates that differences in deuteration upon bindingare the result of a decrease in backbone dynamics in this regionwhen Nef is bound to SH3. The change comes earliest forConsensus G2E Nef.

The major implication of these results for HIV-1 Nef is thatpreviously unrecognized conformational differences may playa role in reduced Nef function. Despite minor sequence varia-tion between the Nef alleles, the analysis of the HX peptidedata suggests that differences in deuterium incorporation arethe result of proteins with slightly different solution conforma-tions and altered binding capacity. It is tempting to speculate asto how the conformational differences seen between these Nefproteins translate into functional differences. In some importantfunctional regions, there were clear differences in deuterium

exchange and, therefore, in conformation/dynamics. Overall,HX reveals that LTNP4 appears to be a more protected, intrin-sically rigid molecule. Whether individual alterations in theamino acid sequence can explain the substantial protectionafforded to LTNP4, or whether it is the sum of all of thesechanges, remains unknown. Perhaps Nef proteins from LTNPsare more conformationally restricted in general and hence lesswell adapted to form the many conformations required for fullbiological activity. A large-scale conformational characteriza-tion of a broad spectrum of Nef alleles byHXMS, including allmajor clades and multiple LTNPs, will help to clarify the roleof conformational changes in impaired LTNP Nef function.

ConclusionsComparing HX MS at the peptide level for related proteins ofvariable sequence is far removed from the much more straight-forward measurement of HX MS in a single protein as wasdescribed by Brian Chait so many years ago. Other consider-ations and the magnitude of their influence will likely not beknown until much more HX MS data for protein families isacquired and analyzed. This example of five Nef proteins is onthe small side of such comparison experiments that are current-ly in progress. The caveats that must be considered for com-parison experiments, especially when making relative deuteri-um measurements, are many and must all be considered. Aswas shown here, certain types of experiments—namely bind-ing experiments—can provide a great deal of information abouthow related proteins behave.

AcknowledgmentsThe authors gratefully acknowledge the NIH AIDS Researchand Reference Reagent Program for providing HIV Nef clones.Financial support fromNIH (to J.R.E.: GM070590, GM086507,GM101135; to T.E.S.: AI102724, AI057083). They thank AmyAndreotti for insightful comments about the data.

References1. Katta, V., Chait, B.T.: Conformational changes in proteins probed by

hydrogen- exchange electrospray-ionization mass spectrometry. RapidCommun. Mass Spectrom. 5, 214–217 (1991)

2. Wales, T.E., Engen, J.R.: Partial unfolding of diverse SH3 domains on awide timescale. J. Mol. Biol. 357, 1592–1604 (2006)

3. Fang, J., Nevin, P., Kairys, V., Venclovas, C., Engen, J.R., Beuning, P.J.:Conformational analysis of processivity clamps in solution demonstratesthat tertiary structure does not correlate with protein dynamics. Structure22, 572–581 (2014)

4. Fackler, O.T., Baur, A.S.: Live and let die: Nef functions beyond HIVreplication. Immunity 16, 493–497 (2002)

5. Greenway, A.L., Holloway, G., McPhee, D.A.: HIV-1 Nef: a critical factorin viral-induced pathogenesis. Adv. Pharmacol. 48, 299–343 (2000)

6. Joseph, A.M., Kumar, M., Mitra, D.: Nef: "necessary and enforcing factor"in HIV infection. Curr. HIV Res. 3, 87–94 (2005)

7. Cullen, B.R.: HIV-1 Nef protein: an invitation to a kill. Nat. Med. 5, 985–986 (1999)

8. Kestler, H.W.D., Ringler, D.J., Mori, K., Panicali, D.L., Sehgal,P.K., Daniel, M.D., Desrosiers, R.C.: Importance of the nef gene


for maintenance of high virus loads and for development of AIDS.Cell 65, 651–662 (1991)

9. Saksena, N.K., Wang, B., Dyer, W.B.: Biological and molecular mecha-nisms in progression and non-progression of HIV disease. AIDS Rev. 3,133–144 (2001)

10. Rodes, B., Toro, C., Paxinos, E., Poveda, E., Martinez-Padial, M., Benito,J.M., Jimenez, V., Wrin, T., Bassani, S., Soriano, V.: Differences in diseaseprogression in a cohort of long-term non-progressors after more than 16years of HIV-1 infection. Aids 18, 1109–1116 (2004)

11. Crotti, A., Neri, F., Corti, D., Ghezzi, S., Heltai, S., Baur, A., Poli, G.,Santagostino, E., Vicenzi, E.: Nef alleles from human immunodeficiencyvirus type 1-infected long-term nonprogressor hemophiliacs with or with-out late disease progression are defective in enhancing virus replication andCD4 down-regulation. J. Virol. 80, 10663–10674 (2006)

12. Tolstrup, M., Laursen, A.L., Gerstoft, J., Pedersen, F.S., Ostergaard, L.,Duch, M.: Cysteine 138 mutation in HIV-1 Nef from patients with delayeddisease progression. Sex. Health 3, 281–286 (2006)

13. Kirchhoff, F., Easterbrook, P.J., Douglas, N., Troop, M., Greenough, T.C.,Weber, J., Carl, S., Sullivan, J.L., Daniels, R.S.: Sequence variations inhuman immunodeficiency virus type 1 Nef are associated with differentstages of disease. J. Virol. 73, 5497–5508 (1999)

14. Carl, S., Greenough, T.C., Krumbiegel, M., Greenberg,M., Skowronski, J.,Sullivan, J.L., Kirchhoff, F.: Modulation of different human immunodefi-ciency virus type 1 Nef functions during progression to AIDS. J. Virol. 75,3657–3665 (2001)

15. Salvi, R., Garbuglia, A.R., Di Caro, A., Pulciani, S., Montella, F.,Benedetto, A.: Grossly defective nef gene sequences in a human immuno-deficiency virus type 1-seropositive long-term nonprogressor. J. Virol. 72,3646–3657 (1998)

16. Deacon, N.J., Tsykin, A., Solomon, A., Smith, K., Ludford-Menting, M., Hooker, D.J., McPhee, D.A., Greenway, A.L., Ellett,A., Chatfield, C., Lawson, V.A., Crowe, S., Maerz, A., Sonza, S.,Learmont, J., Sullivan, J.S., Cunningham, A., Dwyer, D., Dowton,D., Mills, J.: Genomic structure of an attenuated quasi species ofHIV-1 from a blood transfusion donor and recipients. Science 270,988–991 (1995)

17. Hanna, Z., Kay, D.G., Rebai, N., Guimond, A., Jothy, S., Jolicoeur, P.: Nefharbors a major determinant of pathogenicity for an AIDS-like diseaseinduced by HIV-1 in transgenic mice. Cell 95, 163–175 (1998)

18. Geyer, M., Fackler, O.T., Peterlin, B.M.: Structure–function relationshipsin HIV-1 Nef. EMBO Rep. 2, 580–585 (2001)

19. Kirchhoff, F., Schindler, M., Specht, A., Arhel, N., Munch, J.: Role of Nefin primate lentiviral immunopathogenesis. Cell. Mol. Life Sci. 65, 2621–2636 (2008)

20. Grzesiek, S., Bax, A., Clore, M., Gronenborn, A.M., Hu, J.-S., Kaufman, J.,Palmer, I., Stahl, S.J., Wingfield, P.T.: The solution structure of HIV-1 Nefreveals an unexpected fold and permits delineation of the binding surfacefor the SH3 domain of Hck tyrosine protein kinase. Nat. Struct. Biol. 3,340–345 (1996)

21. Lee, C.H., Saksela, K., Mirza, U.A., Chait, B.T., Kuriyan, J.: Crystalstructure of the conserved core of HIV-1 Nef complexed with a Src familySH3 domain. Cell 85, 931–942 (1996)

22. Arold, S., Franken, P., Strub, M.P., Hoh, F., Benichou, S., Benarous, R.,Dumas, C.: The crystal structure of HIV-1 Nef protein bound to the Fynkinase SH3 domain suggests a role for this complex in altered T cellreceptor signaling. Structure 5, 1361–1372 (1997)

23. Grzesiek, S., Bax, A., Hu, J.S., Kaufman, J., Palmer, I., Stahl, S.J., Tjandra,N., Wingfield, P.T.: Refined solution structure and backbone dynamics ofHIV-1 Nef. Protein Sci. 6, 1248–1263 (1997)

24. Geyer, M., Munte, C.E., Schorr, J., Kellner, R., Kalbitzer, H.R.: Structureof the anchor-domain of myristoylated and non-myristoylated HIV-1 Nefprotein. J. Mol. Biol. 289, 123–138 (1999)

25. Barnham, K.J., Monks, S.A., Hinds, M.G., Azad, A.A., Norton, R.S.:Solution structure of a polypeptide from the N terminus of the HIV proteinNef. Biochemistry 36, 5970–5980 (1997)

26. Geyer, M., Peterlin, B.M.: Domain assembly, surface accessibility, andsequence conservation in full length HIV-1 Nef. FEBS Lett. 496, 91–95(2001)

27. Jia, X., Singh, R., Homann, S., Yang, H., Guatelli, J., Xiong, Y.: Structuralbasis of evasion of cellular adaptive immunity by HIV-1 Nef. Nat. Struct.Mol. Biol. 19, 701–706 (2012)

28. Ren, X., Park, S.Y., Bonifacino, J.S., Hurley, J.H.: HowHIV-1 Nef hijacksthe AP-2 clathrin adaptor to downregulate CD4. eLife 3, e01754 (2014)

29. Alvarado, J.J., Tarafdar, S., Yeh, J.I., Smithgall, T.E.: Interaction with theSrc homology (SH3-SH2) region of the Src-family kinase Hck structuresthe HIV-1 Nef dimer for kinase activation and effector recruitment. J. Biol.Chem. 289, 28539–28553 (2014)

30. O'Neill, E., Kuo, L.S., Krisko, J.F., Tomchick, D.R., Garcia, J.V., Foster,J.L.: Dynamic evolution of the human immunodeficiency virus type 1pathogenic factor. Nef. J. Virol. 80, 1311–1320 (2006)

31. Trible, R.P., Emert-Sedlak, L., Wales, T.E., Ayyavoo, V., Engen, J.R.,Smithgall, T.E.: Allosteric loss-of-function mutations in HIV-1 Nef from along-term non-progressor. J. Mol. Biol. 374, 121–129 (2007)

32. Wales, T.E., Engen, J.R.: Hydrogen exchange mass spectrometry for theanalysis of protein dynamics. Mass Spectrom. Rev. 25, 158–170 (2006)

33. Narute, P.S., Smithgall, T.E.: Nef alleles from all major HIV-1 cladesactivate Src-family kinases and enhance HIV-1 replication in an inhibitor-sensitive manner. PLoS One 7, e32561 (2012)

34. Hochrein, J.M., Lerner, E.C., Schiavone, A.P., Smithgall, T.E., Engen, J.R.:An examination of dynamics crosstalk between SH2 and SH3 domains byhydrogen/deuterium exchange and mass spectrometry. Protein Sci. 15, 65–73 (2006)

35. Narute, P.S.: HIV-1 Nef-Src family kinase interaction: a noveltarget for the inhibition of HIV-1 pathogenesis. University of Pitts-burgh: pp 175 (2012)

36. Moarefi, I., LaFevre-Bernt, M., Sicheri, F., Huse, M., Lee, C.H., Kuriyan,J., Miller, W.T.: Activation of the Src-family tyrosine kinase Hck by SH3domain displacement. Nature 385, 650–653 (1997)

37. Engen, J.R.: Analysis of unfolding and protein dynamics in the regulatorydomains of hematopoietic cell kinase with hydrogen exchange and massspectrometry. Doctoral Dissertation, Dept. of Chemistry. University ofNebraska-Lincoln, Lincoln (1999)

38. Mandell, J.G., Baerga-Ortiz, A., Akashi, S., Takio, K., Komives, E.A.:Solvent accessibility of the thrombin-thrombomodulin interface. J. Mol.Biol. 306, 575–589 (2001)

39. Zhang, Z., Smith, D.L.: Determination of amide hydrogen exchange bymass spectrometry: a new tool for protein structure elucidation. Protein Sci.2, 522–531 (1993)

40. Engen, J.R., Smithgall, T.E., Gmeiner, W.H., Smith, D.L.: Identificationand localization of slow, natural, cooperative unfolding in the hematopoi-etic cell kinase SH3 domain by amide hydrogen exchange and massspectrometry. Biochemistry 36, 14384–14391 (1997)

41. Engen, J.R., Wales, T.E., Chen, S., Marzluff, E.M., Hassell, K.M., Weis,D.D., Smithgall, T.E.: Partial cooperative unfolding in proteins as observedby hydrogen exchange mass spectrometry. Int. Rev. Phys. Chem. 32, 96–127 (2013)

42. Wales, T.E., Fadgen, K.E., Gerhardt, G.C., Engen, J.R.: High-speed andhigh-resolution UPLC separation at zero degrees Celsius. Anal. Chem. 80,6815–6820 (2008)

43. Wang, L., Pan, H., Smith, D.L.: Hydrogen exchange-mass spectrometry:optimization of digestion conditions. Mol. Cell. Proteom. 1, 132–138(2002)

44. Wu, Y., Kaveti, S., Engen, J.R.: Extensive deuterium back-exchange incertain immobilized pepsin columns used for H/D exchange mass spec-trometry. Anal. Chem. 78, 1719–1723 (2006)

45. Hochrein, J.M., Wales, T.E., Lerner, E.C., Schiavone, A.P., Smithgall,T.E., Engen, J.R.: Conformational features of the full-length HIV andSIV Nef proteins determined by mass spectrometry. Biochemistry 45,7733–7739 (2006)

46. Pene-Dumitrescu, T., Shu, S.T., Wales, T.E., Alvarado, J.J., Shi, H.,Narute, P., Moroco, J.A., Yeh, J.I., Engen, J.R., Smithgall, T.E.: HIV-1Nef interaction influences the ATP-binding site of the Src-family kinase.Hck. BMC Chem. Biol. 12, 1 (2012)

47. Hvidt, A., Linderstrom-Lang, K.: Exchange of hydrogen atoms in insulinwith deuterium atoms in aqueous solutions. Biochim. Biophys. Acta 14,574–575 (1954)

48. Choi, H.J., Smithgall, T.E.: Conserved residues in the HIV-1 Nef hydro-phobic pocket are essential for recruitment and activation of the Hcktyrosine kinase. J. Mol. Biol. 343, 1255–1268 (2004)

49. Mayne, L.: Hydrogen exchange mass spectrometry. Methods Enzymol.566, 335–356 (2016)

50. Bai, Y., Milne, J.S., Mayne, L., Englander, S.W.: Primary structure effectson peptide group hydrogen exchange. Proteins 17, 75–86 (1993)

51. Molday, R.S., Englander, S.W., Kallen, R.G.: Primary structureeffects on peptide group hydrogen exchange. Biochemistry 11,150–158 (1972)


52. Saksela, K., Cheng, G., Baltimore, D.: Proline-rich (PxxP) motifs in HIV-1Nef bind to SH3 domains of a subset of Src kinases and are required for theenhanced growth of Nef+ viruses but not for down-regulation of CD4.EMBO J. 14, 484–491 (1995)

53. Sheff, J.G., Rey, M., Schriemer, D.C.: Peptide–column interactions andtheir influence on back exchange rates in hydrogen/deuterium exchange-MS. J. Am. Soc. Mass Spectrom. 24, 1006–1015 (2013)


hydrogen exchange mass spectrometry of related proteins … · 2017-08-28 · although much ofthe...

Documents