specificity of the interaction between uba domains and ubiquitin · 2004-01-05 · minimization and...

Specificity of the interaction between UBA domains and ubiquitin

Thomas D. Mueller#, Mariusz Kamionka and Juli Feigon*Department of Chemistry and Biochemistry

405 Hilgard Avenue, P.O. Box 951569

University of California

Los Angeles, CA 90095-1569

*corresponding author; email: [email protected]; phone 310 206 6922; fax 310 825 0982

# present address:

Dept. Physiological Chemistry II

Biocenter, University Wuerzburg

Am Hubland

D-97074 Wuerzburg

Germany

Phone: +49 931 888 4170

Fax: +49 931 888 4113

Email: [email protected]

Running title: Interaction between HHR23A UBA domains and ubiquitin

Submitted 11/24/03; Revised 12/30/03

JBC Papers in Press. Published on January 5, 2004 as Manuscript M312865200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Summary

UBA (ubiquitin associated) domains are found in a large number of proteins with diverse

functions involved in ubiquitination, DNA repair, and signaling pathways. Recent studies have

shown that several UBA domain proteins interact with ubiquitin (Ub), specifically p62, the

phosphotyrosine independent ligand of the SH2 domain of p56lck, HHR23A, a human

nucleotide excision repair protein, and DDI1, another damage inducible protein. NMR chemical

shift mapping reveals that Ub binds specifically but weakly to a conserved hydrophobic epitope

on HHR23A UBA(1) and UBA(2) and that the UBA domains bind on the hydrophobic patch on

the surface of the five-stranded β-sheet of Ub. Models of the UBA(1)-Ub and UBA(2)-Ub

complexes obtained from de novo docking reveal different orientations of the UBA domains on

the Ub surface compared to those obtained by homology modeling with the related CUE

domains, which also bind Ub. Our results suggest that UBA domains may interact with Ub as

well as other proteins in more than one way while utilizing the same binding surface.

Keywords: HHR23A, Rad23, NMR, Ubl, proteasome, structure

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Introduction

The lifespan of proteins inside and outside a cell is tightly regulated by the ubiquitin-proteasome

system, and numerous studies show that protein degradation is tightly interlocked with cell cycle

progression and is therefore an integral part of transduction pathways and other cellular

processes (1-4). For protein degradation by the Ub/proteasome system, the target proteins need

to be tagged with a poly-Ub chain. These covalent complexes are then recognized and degraded

by the 26S proteasome (1,2). The principle mechanism of this covalent modification has been

identified; an enzyme cascade known as E1-E2-E3 is responsible for activation and transfer of

Ub onto the target protein in a linkage specific manner (1,5).

The 26S proteasome is formed by a 20S cylindrical proteolytically active subunit and two

19S regulatory subunits (1,2,6,7). The 19S particles represent the lid of the proteasome and

regulate the access to the proteolysis (8). Although the polyubiquitinated substrate seems to be

recognized by the S5a subunit in the 19S particle (9-11), additional contacts between poly-Ub

chains and parts of the 19S regulatory subunit have been identified (12). Deletion studies indicate

that other polyubiquitin binding sites must exist (13).

Monoubiquitination is not sufficient for targeting proteins to the proteasome, however;

assembly of a poly-Ub chain of at least four Ub moieties is required to create a degradation

signal (11,14,15). Although Ub contains seven lysine residues, they are not used with the same

frequency in poly-Ub chain assembly. The predominant linkages observed are Lys 48-Gly 76

(16), Lys 29-Gly 76 (17) and Lys 63-Gly 76 (18), of which Lys 48-linked chains appear to be

the most frequent degradation signal. Poly-Ub assembly via Lys 29 and Lys 63 is less common,

and chain formation via Lys 63 seems to be involved in non-degradation signal events, e.g.

DNA repair (18).

Although key steps of Ub activation and transfer to a substrate as well as the structure of

the 20S subunit of the proteasome are known, the question of how proteins are targeted to the

proteasome remains unanswered. It is not known if there is an additional mechanism to regulate

the time point of degradation. One possibility is that monoubiquitination leads to a "point of no

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return", which proceeds to substrate destruction in a defined time span. Recently, several groups

reported that proteins containing a UBA motif can bind directly to Ub and/or poly-Ub, leading

to an inhibition of the degradation of target substrates through the proteasome (19-21). UBA

domains are a common motif in a variety of protein families involved in protein degradation, cell

cycle control, or DNA repair. In vitro and in vivo assays have revealed that UBA domains of the

DNA-damage inducible proteins, RAD23 (as well as the fission yeast homologue Rhp23) and

DDI1, as well as proteins with no function in DNA repair, p62 and Mud1p, interact specifically

with Ub. Furthermore, poly-Ub chain formation is inhibited by RAD23 in vitro in a

concentration dependent manner (20,21). In addition, it has been shown that poly-Ub chain

extension stops at a length of three Ub moieties and that the inhibition of chain extension of

RAD23 is specific for Lys48-linked chains (22). As a consequence of the UBA-Ub interaction,

Clarke and co-workers showed that RAD23 and DDI1 are involved in the checkpoint control of

the cell cycle (23). Binding of the UBA domains of RAD23 and DDI1 to the nascent poly-Ub

chain of the Pds1 substrate leads to inhibition of chain extension, which subsequently results in

an increased lifetime of Pds1, which would otherwise be rapidly degraded.

In this report we provide a structural basis for the interaction of UBA domains with

monomeric Ub based on an NMR chemical shift mapping study as well as Ub mutagenesis. Ub

binds specifically to both UBA(1) and UBA(2) of HHR23A. The binding interface for both UBA

domains is almost identical despite the low overall sequence similarity. Both UBA domains bind

to the same region of monomeric Ub, which is also involved in the binding to the proteasome

subunit S5a. Models for the UBA(1)-Ub and UBA(2)-Ub complexes were generated from the

chemical shift mapping data by de novo docking as well as by homology modeling with the

solution structure of the closely related CUE domain in complex with Ub (24). These models

revealed very different orientations of the UBA domains on the surface of Ub. Our results

suggest that UBA domains may interact with Ub as well as other proteins, e.g. the HHR23A

binding proteins HIV-1 Vpr (25), methyladenine DNA glycosylase (MPG) (26), p300/cyclic

AMP responsive element binding (CREB)-protein (27), and peptide:N-glycanase (Png1) (28),

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in more than one way while utilizing the same binding surface.

Experimental proceduresPreparation of proteins

UBA(1) and UBA(2) of HHR23A were prepared as previously described (29,30). 15N-

and 15N-,13C-labeled proteins were prepared by growing cells on M9 minimal medium using

15NH4Cl and 13C6-glucose as the sole nitrogen and carbon sources. For the titration of the UBA

domain proteins bovine Ub (amino acid sequence is identical to human Ub) was purchased from

Sigma and purified by gel filtration. Purity was subsequently checked by SDS-PAGE and

analytical reversed phase HPLC. For the NMR chemical shift mapping of Ub, yeast Ub was

prepared from the expression plasmid pET11c-Ub (gift from A. Varshavsky). The protein was

purified by reversed phase HPLC. Alanine mutants of yeast Ub were generated by site directed

mutagenesis using QuikChange (Stratagene). The cDNA sequence was verified by sequencing

using the DyeDeoxy-Terminator method (PerkinElmer). Ub mutant proteins were prepared

similar to wild type Ub. Protein concentrations were determined using an extinction coefficient

at A280 of 1280 M-1cm-1 for Ub and UBA(2) and 5120 M-1cm-1 for UBA(1), based on their

respective amino acid compositions.

Chemical shift mapping experiments

Proteins for titration experiments were dialyzed against identical buffer (50mM sodium

phosphate pH 6.5, 100mM sodium chloride, 2mM DTT-d10 (Cambridge Isotopes Labeling CIL)

to avoid chemical shift changes due to differences in buffer conditions. NMR samples for

titration studies contained 0.25 to 0.5 mM 15N-labeled protein. Unlabeled protein was added

stepwise up to a final ratio of 1:10. Unlabeled Ub was concentrated as far as possible to

minimize volume changes throughout the titration. The “reverse” titration of Ub with UBA

proteins was performed similar to the experiment described above. A similar mapping study

using 15N-labeled UBA(1) or UBA(2) and the Ubl domain of HHR23A was performed in order

to test the specificity of the UBA domains for Ub. In addition, the UBA domains were tested for

homo- and heterodimerization with NMR mapping experiments. 15N-labeled UBA(1) (0.7mM)

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was mixed with unlabeled UBA(2). All measurements were performed at 27° C using a Bruker

DRX-500 or DRX-600. 2D 1H-15N HSQC experiments with watergate water suppression and

water flip-back pulses were used for monitoring the chemical shift changes. All titration data

sets were processed identical using the software XWINNMR (Bruker). 1H dimensions were

referenced using external TSP. 15N nitrogen and 13C carbon frequencies were referenced using

the gyromagnetic ratios 15N/1H = 0.101329118 and 13C/1H = 0.251449530. The changes of

proton and nitrogen chemical shifts were averaged based on their gyromagnetic ratios using the

following equation: ∆δave. = 0.5x [(|∆δ(1H)|+(0.125x|∆δ(15N)|)].

The chemical shifts of yeast Ub were reassigned (chemical shifts published for human Ub

differ from yeast Ub due to three amino acid changes) using 15N,13C-labeled yeast Ub. A set of

triple-resonance experiments (31) (CBCA(CO)NH, CBCANH, HBHA(CO)NH, H(C)(CO)NH-

TOCSY, CC(CO)NH-TOCSY, HCCH-COSY) was acquired to assign all backbone and side-

chain chemical shifts for yeast Ub under the conditions used for the NMR chemical shift

perturbation studies.

Homology modeling of the UBA-ubiquitin complexes

Model complexes of the UBA(1)/UBA(2) with Ub were built using the structure of the

CUE domain of Cue2 and Ub as a template (24). Initial structures of the complexes were

obtained by fitting the Cα-atoms of the residues in helices 1 and 3 of the CUE and UBA

domains. Close contacts between atoms in the interface were removed by manual remodeling

using the software Quanta98. The structures were then subsequently refined by energy

minimization and short (20 ps) molecular dynamics simulation at 300K (Charmm force field,

Quanta98) using only geometrical energy terms to maintain bond length and angles as well as

van der Waals packing.

De novo docking for the UBA-ubiquitin interaction

The program HADDOCK (32) was applied to dock Ub and the UBA domains of

HHR23A. In this approach, data obtained from chemical shift mapping is transformed into a set

of ambiguous distance restraints that are used together with geometrical and electrostatic

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complementarity to dock the two molecules. Chemical shift mapping of UBA(1), UBA(2), and

Ub and surface accessibility data calculated with the program NACCESS (33) were used to

define ambiguous distance restraints as described. The target distance of these restraints was set

to 2.0 Å; force constants for empirical and experimental restraints were used as suggested by the

default settings. Initially 750 structures for a Ub-UBA complex were generated by docking Ub

and UBA(1)/UBA(2) as rigid bodies using only ambiguous distance restraints, van der Waals

energy and electrostatic energy terms using the program suite ARIA1.2 and CNS (34,35). Of

those, 200 structures with the lowest overall energy were subsequently refined allowing the side

chain conformations of residues within the binding interface to be flexible. Finally, 100 refined

structures with lowest overall energy were then refined using a short molecular dynamics

simulation in explicit solvent to allow for a correct implementation of the electrostatic

contribution. The method was applied to two closely related complexes for which structures were

available, the UIM-2 S5a-HHR23A Ubl and the Cue2 CUE domain-Ub complex, to test for the

reliability of that procedure. For the simulation of the Cue2 CUE domain-Ub interaction, the

distance restraint set was defined based on the residues buried in the complex structure; for the

simulation of the HHR23A Ubl-UIM-2 S5a interaction the same criteria for chemical shift

changes and residue accessibility as used for the Ub-UBA domain modeling were applied for

generating the distance restraint set. The test calculations yielded model structures that were

within 1.5 Å rmsd of the experimentally determined structures. Different cutoff values for the

surface accessibility in the definition of the distance restraint set used for the computational

docking did not lead to altered complex architectures, confirming the computational results are

stable. The influence of the template structures of the individual complex components was

determined by using the Ub structure of the Cue2 CUE-Ub complex for docking to the UBA

domains as well as the structure of free Ub (PDB entry code 1UBI). Again no change in complex

architecture could be observed.

Results

Ubiquitin binds specifically to HHR23A UBA(1) and UBA(2) domains

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High-resolution structures of both UBA domains of HHR23A have been determined,

which show that overall the three-helical bundles are largely identical, in spite of a low (~20%)

sequence identity (29,30,36). The main structural differences are slightly different packing of

side chains in the hydrophobic core and the conformation of the N- and C-termini. Both

domains have an unusually large hydrophobic surface patch which was predicted to be a protein-

protein interface (29,30). Based on the structures and sequence analysis of UBA domains, we

predicted that a conserved portion of this hydrophobic patch would be the binding region for Ub

(29).

In order to map the binding site of Ub on UBA domains, NMR chemical shift

perturbation experiments were performed on both UBA(1) and UBA(2) from HHR23A. Samples

of 15N-labeled UBA(1) and UBA(2) were mixed with bovine Ub, and 2D HSQC spectra were

acquired to monitor the changes in the chemical shifts of the backbone amides induced by the

binding to Ub (Fig. 1 A, B). Changes in chemical shifts are observed for several residues in both

UBA(1) and UBA(2) starting at about 0.6 molar equivalents, indicating the formation of specific

complexes in fast exchange on both the 500 and 600 MHz NMR time scale. Assuming a binary

interaction between the UBA domains and Ub, a KD in the range of 500 to 600 µM can be

determined from non-linear fitting of the titrations with either UBA(1) or UBA(2) (see also Fig.

3A, B). However, a binding constant of about 10 µM was reported for full-length Rad23 (19),

which may indicate cooperative binding of both UBA domains (37). Plots of the chemical shift

changes versus residue (Fig. 1 C, D) show that the absolute magnitude of the chemical shift

changes are similar for UBA(1) and UBA(2).

A recent chemical shift mapping study of the closely related HHR23B reported that the

Ubl and UBA domains interact with each other with a reported KD of ~2 mM, which is ~10-fold

higher than their calculated KD for the HHR23B-UBA interactions of ~300 µM (38). The Ubl

domain is structurally very similar to Ub (39,40) and exhibits a high sequence identity. Weak

binding between the HHR23A Ubl and UBA domains was also detected in the context of the full

length protein as well as isolated domains (41) under slightly different buffer conditions and at

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higher field strength, with a binding affinity at least 10-fold lower compared to the already weak

interaction between the UBA domains and Ub.

Ubiquitin binds to a conserved surface epitope of UBA domains

The results of the chemical shift perturbation study were mapped onto the structures of

UBA(1) and UBA(2) (Fig. 1). Although UBA(1) and UBA(2) have a relatively low sequence

identity, the binding interfaces revealed by the chemical shift mapping are remarkably similar.

Residues exhibiting the largest chemical shift changes upon binding to Ub, G174 and H192 for

UBA(1) and L330, G331 and E348 for UBA(2), respectively, are in similar locations. A large

cluster of residues in UBA(1) involved in binding is located at the C-terminal end of the first

helix, I170, M171, S172 and the first three residues in the short and highly conserved loop 1,

M173, G174 and Y175 (Fig. 1 A, C, E). Several positions on the third helix of UBA(1) also

show large changes upon binding, specifically H192 and R193 at the N-terminus of helix 3 and

Y197 at the second turn of helix 3. Together, these residues form a consecutive patch of about

520 Å on the surface of UBA(1) (Fig. 1 E). The residues with the largest chemical shift changes,

H192, M173, and G174, are in the center of the epitope.

Residue Y197 also exhibits a significant change in chemical shift upon addition of Ub,

but is located on the “back face” of the binding interface. The change for the amide proton and

nitrogen frequencies of Y197 might be attributed to small structural changes in the hydrophobic

core due to interactions of the side chain of Y197 with R193, which is part of the binding

interface. Residues in the C-terminus of UBA(1) do not exhibit chemical shift changes when Ub

is added (Fig. 1 C) and do not seem to be part of the binding interface. This was surprising, since

the C-terminus is close to the hydrophobic patch involved in binding and is of relatively rigid

nature (29).

Analysis of the binding of UBA(2) to Ub reveals that the general location of the epitope

remains the same compared to UBA(1) (Fig. 1). Identical positions in the C-terminal end of

helix 1 (R326, L327 and A329) as well as the first three residues of the hydrophobic loop 1

(L330, G331 and F332) form a large part of the binding interface to Ub. In addition, residues at

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similar positions at the N-terminus of helix 3 (E348 and N349) are also among the residues with

the highest chemical shift changes upon binding. The structurally equivalent residue of E348 in

UBA(1) is P191, therefore no information about whether or not P191 is involved in the binding

of Ub can be deduced from this analysis. A larger number of residues at the C-terminal end of

helix 3 of UBA(2) are involved in binding than observed for UBA(1). On the last turn of helix 3

residue L356 is affected by the binding, while residues of UBA(1), L199 and T200, do not

exhibit significant changes. Overall, with a binding area of approximately 650 Å2, UBA(2)

appears to have about a 25% larger interface than UBA(1), for which the residues within the

binding epitope yield an area of about 520 Å2.

UBA domains bind on the 5-stranded β-sheet surface of ubiquitin

In order to investigate the binding surface for UBA domains on Ub, NMR chemical shift

mapping was performed under the identical conditions as reported above using yeast Ub. Yeast

Ub differs in three amino acids compared to human Ub, S21P, D24E, and S28A, none of which

is close to the binding site for UBA domains determined in this study. The residue P19 in human

Ub is located in a tight turn in the loop between the second β-strand and the first α-helix. The

two other residues D24 and S28 are located on the α↑helix, facing in the opposite direction of

the determined binding interface. We therefore concluded that these mutations do not interfere

with or modulate the binding of the UBA domains to Ub. However, to confirm that the

recognition process is not influenced by indirect effects we repeated the titration using 15N-

labeled human Ub purchased from VLI research (Mavern) and UBA(2); no differences in the

chemical shift changes compared to the study using yeast Ub were detected (data not shown).

Chemical shift mapping of the amide resonances of Ub as a function of added UBA(1) or

UBA(2) up to a ratio of 1:10 Ub:UBA again revealed complexes in fast exchange on the NMR

timescale. Upon addition of UBA(1) to Ub significant changes in chemical shift are observed for

23 residues (Fig. 2 A, C, E). Most of these residues are located on the β-strands or the

connecting loops of the 5-stranded β-sheet of Ub, forming a consecutive patch. L71 to L73 are

located in the C-terminus close to the Gly-Gly motif required for poly-Ub chain extension (Fig.

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2 E). When Ub was titrated with UBA(2), equivalent residues showing a significant change in

their proton and nitrogen amide frequencies were almost identical with those for UBA(1) (Fig.

2).

These results suggest that both UBA domains are recognized by and bind to Ub using a

similar binding epitope. The binding epitopes of UBA(1) and UBA(2) on Ub overlap perfectly,

and the differences in several of the amino acids, e.g. H192 versus E348 and R193 versus N349,

might account for the chemical shift differences observed for the titration of Ub with either

UBA(1) or UBA(2). However, another possibility is that the two UBA domains are oriented

differently on Ub, as discussed below.

Differential chemical shift mapping of UBA domains

To further address the question how the UBA domains of HHR23A bind to Ub,

especially whether the binding mechanism differs between the two UBA domains, we tried

differential chemical shift mapping (42). Five Ub mutants (L8A, R42A, K48A, H68A, and

R72A) with single amino acid substitutions were chosen for study. All of the mutated residues

have side chains oriented towards the binding interface and show significant chemical shift

changes upon binding to the UBA domains. Differences in the chemical shift changes between a

Ub mutant and wild type Ub should be observed if the residue pair is in close proximity in the

UBA-Ub complex. The binding affinities were determined by non-linear fitting of the chemical

shift changes of three residues located within the binding epitope (I170, G174, and H192 for

UBA(1); G331, E348, and L356 for UBA(2)).

Surprisingly, the mutant Ub L8A exhibits much smaller absolute chemical shift changes

upon binding to UBA(1) and UBA(2), suggesting a lower binding affinity. However, quantitative

analysis of the binding curves (Fig. 3A,B) shows that the affinity of UBA(1) and Ub L8A is not

changed significantly (490µM and 570µM, respectively). Similar small changes in KD (less than

2-fold) were observed for the other Ub mutants (K48A, H68A, R72A). Interestingly, the binding

affinity of the Ub mutant R42A for UBA(1) and UBA(2) seems to be increased 3.5- and 2-fold .

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Comparison of the UBA-Ub titration studies with those of the Ub mutants showed no

significant differences in the course of the chemical shift changes for UBA(2). In contrast, for

UBA(1), the chemical shift pattern of Ub mutants L8A and R42A is different compared with

wild type Ub. For L8A significant changes are observed for residues E169 in helix 1 as well as

R193, A194, and E196 in helix 3 (Fig. 3C) indicating that these residues are in close proximity to

each other in the complex. For Ub mutant R42A, the largest differences are observed for residues

E169 (helix 1), and A194, E196, L199 and T200 located in helix 3 (Fig. 3D). Since the side

chains of L8 and R42 are separated by only 6 Å, similar residues can be affected by both Ub

mutants. Model building of a UBA(1)-Ub complex based on just the two identified residue pairs

is however not possible since two interaction points do not define the orientation of two rigid

bodies unambiguously.

These mutagenesis results are consistent with an analysis of the interaction of the CUE

domain of Cue2 protein using two Ub mutants K48A and H68A (24). Although both mutations

are located in the center of the interacting patch they did not influence the interaction

significantly. Apparently, the binding specificity between Ub and its interaction partners is

achieved by the overall surface topology, which explains why a single point mutation is not able

to destabilize the binding substantially.

Homology modeling of the UBA-Ub interaction

Recently, the structures of a UBA homolog the CUE domain in complex with Ub was

determined by NMR and X-ray crystallography (24,43). Based on the structure of the CUE

domain of Cue2 bound to Ub (24), we generated a model for the interaction of UBA(1) and

UBA(2) with Ub (Fig. 4A, B). The solution structure of the Cue2-Ub complex instead of the

crystal structure of the CUE domain of Vps9 bound to Ub (43) was used for model building due

to the higher similarity between the CUE domain structure of Cue2 and the UBA domains of

HHR23A. The crystal structure of the CUE domain of Vps9p forms a domain-swapped inter-

twined dimer and binding to Ub results in a large conformational change. Furthermore, the

binding affinity of the Cue2 CUE domain to Ub (KD ~150µM) is more similar to the interaction

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of the UBA domains of HHR23A with Ub (KD ~500µM). In contrast the binding of the Vps9p

CUE domain is considerably tighter (KD ~ 20µM). The rmsd for Cα positions of the three

helices of UBA and CUE are 1.6 Å (CUE - UBA(2), 29 Cα positions) and 1.9 Å (CUE -

UBA(1), 26 Cα positions). In the final models 410 Å2 (UBA(1)-Ub) and 440 Å2 (UBA(2)-Ub)

surface area of the UBA domains are buried in the interface. This is in good agreement with the

interface size determined by chemical shift mapping. The nature of the interface is almost

exclusively hydrophobic (>80%).

In the UBA-Ub models the axis of helix 1 is oriented in an angle of 45° to the β-strand 5

of Ub, helix 3 is running at an angle of 50° across the β5, the helical axis of helix 2 is running

almost in parallel with β5 (Fig. 4A). Hydrophobic residues in loop 1 (G174-Y175 UBA(1) and

G331-F332 UBA(2)) of the UBA domain interact with the β3-β4 loop residues of Ub (Fig. 4A).

The N-terminus of helix 3 of the UBA domains (P191-H192 UBA(1) and E348-N349 UBA(2))

is close to the C-terminus of β-strand 5 and the β1-β2 loop of Ub. The important hydrophobic

triad of Ub, residues L8, I44, and V70, is buried in the interface. In the Ub-UBA(1) complex, L8

of Ub is surrounded by UBA(1) residues E169, I170 (helix 1), P191, and H192 (loop 2) (Fig.

4A). In the complex of Ub-UBA(2), residue L8 of Ub has van der Waals contacts to residues of

UBA(2) occupying similar positions in the three-helical bundle (A323, L327 (helix 1), and E348

(loop 2)) (see Fig. 4B). I44 of Ub is packed against M173 (helix 1), Y175 (loop 1), and V195

(helix 3) of UBA(1); in Ub-UBA(2) residues L330 (helix 1), A352, and L356 (helix 3) of

UBA(2) are close to I44 of Ub. V70 of Ub either contacts H192 (loop 2) of UBA(1), or residues

A323, L327 (helix 1) and E348 (loop2) of UBA(2).

The differences in the environment of Ub V70 bound to UBA(1) vs. UBA(2) are due to

slightly different interhelical angles of the three-helical bundle, which lead to helix 1 of UBA(1)

pointing further away from the binding interface. Although different residues of both UBA

domains interact with L8, I44, and V70 of Ub, the hydrophobic nature in the contact area is

preserved. Only a very small number of possible intermolecular hydrogen bonds can be

identified in the binding interfaces of the model complexes: 5 for Ub-UBA(1) (Ub-UBA: K6-

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E169, G47-Y175, G47-M173, H68-E169, L71-H192) and 2 for Ub-UBA(2) (Ub-UBA: T7-

R326, H68-A329). Similarly, in the complex of Cue2 CUE domain and Ub only two hydrogen

bonds are observed. The dearth of polar interactions in addition to the relatively small interface

area (~500 Å2) might explain the relatively low binding affinity between the monomeric CUE

domain and Ub as well as for UBA domains and Ub.

De novo docking of the ubiquitin-UBA domain complexes

In addition to the homology modeling, we performed a de novo docking using the

program HADDOCK and employing data from our chemical shift mapping (32). The chemical

shift changes of UBA(1), UBA(2), and Ub were used together with surface accessibility data to

define ambiguous distance restraints between the two molecules. The method relies on

geometrical and electrostatic complementarity of the binding epitopes of the interacting

molecules, and it has been applied successfully to other complexes (32). Much to our surprise,

the results of the de novo docking of the Ub-UBA domain complexes revealed completely

different complex structures compared to those obtained by homology modeling (Fig. 4 and 5).

Additionally, the architecture of the complexes resulting from de novo docking for the Ub-

UBA(1) and Ub-UBA(2) interaction (Fig. 5 A,B) is also different, indicating that the UBA

domains of HHR23A might interact with Ub differently.

A structural alignment of the Ub molecules of the two models of UBA(2)-Ub (homology

model vs. de novo docking) shows the large difference. In the complex of UBA(2)-Ub obtained

by de novo docking, helices 1 and 3 of UBA(2) run almost parallel to β-strand 5 of Ub, and

UBA(2) helix 2 and Ub β-strand 5 are oriented at an angle of about 20° (Fig. 5B) relative to

each other. In the homology model (and hence for the template complex Cue2 CUE domain-Ub)

,helices 1 and 3 of the UBA domain and β-strand 5 of Ub share an angle of almost 45°; and the

helical axis of helix 2 of UBA(2) runs parallel to β-strand 5 of Ub (Fig. 4B). Consequently, the

de novo docking model can be transformed into the homology model by a rotation of about 45°

in counter clockwise direction. A large positional movement is observed for the residues in the

GFP-loop of UBA(2), with the Cα-positions of L330, G331, and F332 differing by 7 to 8 Å

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between the two modeling approaches. The positional change of the residues in the loop 2 (E348

and N349) and helix 3 (L356) with respect to the Ub binding interface is smaller (distances for

Cα positions: E348 3 Å; N349 4 Å, and L356 4 Å). Consequently a different residue pairing is

observed in the interface of the de novo docking model. L8 of Ub is surrounded by R326, L327,

L330, and E348 of UBA(2), I44 of Ub is in van der Waals contacts with residues A352, N353,

and L356 of UBA(2) and V70 of Ub is in close proximity to N349 and A352 of UBA(2). In the

complex of the Cue2 CUE domain and Ub (and thus in the homology model of UBA(2)-Ub), the

residues located at the N-terminus of the helix 1 are part of the binding interface, which is not

the case for the de novo docking. In the homology model, A323 and R326 both contact residues

L8 and T9 of Ub, whereas for the de novo docking model, A323 shares no contacts with any

residues of Ub.

De novo docking of UBA(1) and Ub resulted in a model complex that differs from the

homology model to an even greater extent. The helical axes of helix 1, 2, and 3 of UBA(1) in the

two models differ by about 55°, 70°, and 100°, respectively. In order to transform both models

into each other a rotation of almost 90° is required. In the UBA(2)-Ub models the largest

positional discrepancies between the two different models are observed for the GFP-loop, while

for the UBA(1)-Ub models the major positional changes are for helix 3. Residue H192 is located

on top of the β1-β2 loop of Ub and contacts K6, L8, and G10, whereas in the homology model

H192 is positioned between the β-strands 5 and 3 and contacts residues R42, V70, and L71. This

clearly shows that the residue paring is quite different for these two models. In both models the

side chain of M173 of UBA(1) makes comparable contacts and is located in a hydrophobic

pocket formed by H68 and I44 of Ub. In the de novo docking model Y175 of UBA(1) is in close

proximity to R42 of Ub possibly making hydrogen bonds between the hydroxyl group of Y175

and the guanidinium group of R42. In contrast in the homology model Y175 is in van der Waals

contact with G47 of Ub with a possible hydrogen bond between the hydroxyl group Y175 and

the backbone carbonyl of G47 (Fig. 5B). Analysis of the differences between UBA(1)-Ub and

UBA(2)-Ub obtained by de novo docking suggests that the residue pairing might be quite

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different although the residue positions in the UBA domain architecture involved in the binding

are conserved.

Since the results of the de novo docking for UBA(1)-Ub and UBA(2)-Ub deviate from

the models obtained by homology modeling, we tested whether the differences might be an

artifact of the docking procedure. Since all 100 refined structures of both complexes cluster into

one single structure family exhibiting an rmsd within the cluster of less than 0.8 Å, the observed

complex architecture obtained by the docking simulation seems to be very stable. Different sets

of distance restraints for the docking did not lead to altered complex architectures. We also tested

the docking for two other complexes, the Cue2 CUE domain Ub interaction (24) and the

complex of HHR23A Ubl domain and UIM-2 of S5a (39). For both simulations, structure data,

and for the latter, structure and chemical shift mapping data, were available. The docking of the

Cue2 CUE domain and Ub reproduced the experimental structure of Kang et al. (24) with an

rmsd of less than 1 Å for the Cα atoms and about 1.3 Å for all heavy atoms. In the docking

simulation of S5a UIM-2 and the Ubl domain of HHR23A, the final structures obtained had an

rmsd of 1.1 Å and 1.8 Å for the Cα-atoms and all heavy atoms, respectively, compared with the

experimental structure. These simulations confirm that the setup is able to reproduce structures

of two experimentally determined complexes that have similar binding mechanism and chemistry

(mainly hydrophobic interaction).

Discussion

Is the hydrophobic knob a general ubiquitin binding site for UBA domains?

The chemical shift mapping study presented here reveals a structural basis for the

recognition and binding of UBA domains of HHR23A to Ub. The hydrophobic surface patches

that were predicted to be the site of protein-protein interactions for the UBA domains (29)

comprise a large portion of the binding epitope for Ub. In addition, some charged and polar

residues appear to be important based on the chemical shift mapping. These results agree very

well with a mapping study of HHR23B UBA domains published very recently by Choi and

coworkers (38), except for the equivalent residue to HHR23A UBA(1) 197. Despite the

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relatively low binding affinity of 500 µM, the interaction of the UBA domains with Ub is

specific. Furthermore, binding between HHR23A/B UBA and Ubl domains, is an order of

magnitude weaker (38,41).

The amino acid sequence identity of UBA domains is about 25%, which is too low to

propose a common biological function or even a common binding mechanism. However, if the

analysis is limited just to the binding interface determined in this study, the degree of similarity

of the binding epitope is as high as for the hydrophobic core of the UBA domains (Fig. 6). The

binding epitope of Ub on UBA(1) and UBA(2) is comprised of 9 and 12 residues, respectively.

The high degree of conservation for the residues located on the surface (similarity for binding

region >50%, similarity within hydrophobic core ~60%) suggests that these are involved in a

common binding interface (29). The chemical shift mapping study of UBA(1) and UBA(2)

presented here confirms the prediction that these residues in the hydrophobic patch are part of a

binding epitope to Ub. Despite a similar location on UBA(1) and UBA(2), however, we also note

differences in these epitopes. In UBA(2) the second leucine residue L356 of the highly conserved

double leucine motif clearly exhibits changes in its chemical shift upon binding to Ub, whereas

the same residue position in UBA(1), L199, is not affected. In addition, the number of residues

on helix 3 affected in the titration with Ub is different, with six residues of UBA(2) involved in

the binding epitope but only three residues of UBA(1).

The five-stranded β-sheet of ubiquitin is an universal binding site for ubiquitin-interacting

proteins

Despite the differences in the binding epitopes determined for the UBA domains of

HHR23A, the position of the interface on Ub is practically identical for both UBA domains. This

region has been identified for the interaction with several other protein domains indicating that

the hydrophobic surface patch on the five-stranded β-sheet of Ub is probably a general protein-

protein interface that can facilitate various interactions. At least six ubiquitin-interacting

domains have been described so far (44). The structures of five domains have been determined

and the site of their interaction with Ub has been mapped. The three-dimensional structures of

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these domains, UEV (ubiquitin E2 enzyme variant) (45), NZF (novel zinc finger domain) (46),

UIM (ubiquitin-interacting motif) (39,47,48), UBA (29,30,36), and CUE (24,43), vary greatly in

architecture and size, being either a single helix (UIM), pure β-strand structure (NFZ), three

helical bundles (CUE, UBA) or mixed alpha-beta structure (UEV). Despite this large variety, all

domains interact with Ub via the same hydrophobic patch on the five-stranded β-sheet of Ub.

Comparing the binding to Ub for all domains reveals only very minor differences in the location

of the binding sites. The center of the binding site, the hydrophobic patch around residue I44 of

Ub, seems to be identical for all ubiquitin-interacting domains so far, although residues close to

that patch have been mapped to different biological functions (49). Very little structural data for

the interaction of monomeric Ub or ubiquitin-like domains in complex with an ubiquitin-

interacting domain is available so far (24,39,43), probably due to the low to moderate binding

affinities for such interactions (KD ~10 to 500µM) (44). A comparison of the binding mechanism

of a single α-helix with an Ub-like domain (complex of HHR23A Ubl-UIM-2 of S5a) with

that of a three-helical bundle bound to Ub (complexes of Cue2 CUE domain and Vps9p CUE

with Ub) shows that complexes of Ub and Ub-interacting domains can adopt different

architectures. The single α-helix of the UIM motif of S5a binds on top of β-strand 5 of

HHR23A Ubl, with the axes of the α-helix of the UIM motif and of β-strand 5 of Ub running

anti-parallel (39). In contrast, the three-helical bundle of the CUE domain of Cue2 binds via

helices 1 and 3, which run across β-strands 1, 3, and 5 at an angle of about 30° (24). In the case

of the CUE domain of Vps9p, the interaction is even more complex. In the X-ray crystal

structure of the complex of the CUE domain of Vps9p with Ub, the CUE domain forms a

domain-swapped dimer, in which helices 1 and 3 interact with Ub in a similar manner to that

observed for the Cue2 CUE domain-Ub complex, but here helix 2 has additional contacts with

residues of Ub (43). Despite the differences in the architecture of the complexes, the interacting

amino acids are conserved, with only hydrophobic amino acids taking part in the interaction. The

absence of hydrogen bonds in the center of the interface probably explains the limited specificity

of Ub, since there is no requirement to maintain the geometry of hydrogen bonding acceptors or

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donors, and therefore specificity for a binding partner is only generated by geometrical

restrictions for the interacting hydrophobic side chains. It is also interesting to note, that the

binding affinity between Ub and Ub-interacting domains of known structures correlates with the

size of the interface. The Cue2 CUE domain Ub interface measures 450 Å2 and has a binding

affinity of about 150 µM (24), while the S5a UIM-2 HHR23A Ubl complex has a buried surface

area of roughly 600 Å2 (39) and a KD ~10 µM. The interface between the CUE domain of

Vps9p and Ub measures 520 Å2 (due to additional interacting residues in the second helix)

resulting in an affinity of 20 µM (43).

A model for the interaction of UBA domains and ubiquitin

One very surprising result of the modeling of the UBA-Ub interaction presented in this

study is the large differences between the models obtained by homology modeling and de novo

docking using the program HADDOCK. The homology models were built using the NMR

structure of the Cue2 CUE-ubiquitin complex, with the CUE domain being replaced by the UBA

domains of HHR23A. Residues of the Cue2 CUE domain that interact with residues of the five-

stranded β-sheet of Ub are either conserved or replaced by homologous amino acids (Fig. 6) in

the UBA domains of HHR23A. However, docking of UBA(2) to Ub using the program

HADDOCK resulted in a model with the three-helix bundle rotated clockwise by about 45°. For

the de novo docked complex involving UBA(1) the three-helix bundle is rotated by almost 90°

but in counterclockwise direction (Fig. 4 and 5). Thus, not only do the de novo docking results

differ from the homology modeling approach, but the de novo docking even suggests different

complex architectures for UBA(1) and UBA(2) bound to Ub. The differences in the complex of

the two CUE domains of Cue2 and Vps9p with Ub provide experimental support for the idea that

the interface of Ub allows for the binding of different helical bundle geometries. The CUE

domain of Vps9p, being a domain-swapped dimer, binds to Ub not only via helices 1 and 3, but

also via additional residues in helix 2 which likely contribute to affinity and specificity (43).

We note, in addition, that the differential chemical shift mapping employing Ub mutants

showed differences for UBA(1) and UBA(2). These might indicate that both UBA domains bind

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to Ub by a different binding mechanism, supporting the results obtained by the de novo docking

procedure. Analysis of the distribution of charged residues surrounding the hydrophobic patch,

which is in the center of the binding interface for CUE and UBA domains, clearly shows that the

electrostatic potentials are distinct for these domains. Biological data suggest differences in

functions of the CUE and UBA domain (50-52), and sequence comparison clearly distinguishes

between the CUE and UBA family (53). All CUE domains identified so far bind to monomeric

Ub with low to moderate affinity (20 to 160 µM), and some but not all CUE domains seem to

bind to poly-Ub in addition (52). It was not reported whether their binding affinity for poly-Ub

is higher than for monomeric Ub; however data from Shih et al. (52) suggests that at least the

CUE domain of Vps9 has no preference for long poly-Ub chains over short poly-Ub chains.

This binding preference is probably required for the maintenance of monoubiquitination (52),

which is in turn a signal for trafficking and receptor endocytosis. However for a more

quantitative analysis the binding constants of several CUE domains for mono- and poly-Ub

have to be determined.

The biological function of the UBA domain is, on the other hand, still in debate

(20,22,23,37,54). Several groups have reported that UBA domains bind to monomeric Ub as well

as to poly-Ub, although the data is contradictory in some cases (19,54). Binding to monomeric

Ub was associated with inhibition of further extension of the nascent Ub chain which results in

an inhibition of the degradation of a substrate (20-23). The binding to poly-Ub was explained

with a possible shuttle function for the transport of a substrate to the proteasome (37,54-57).

Although the physiological “Ub-target” of the UBA domains of RAD23 and other proteins has

not been determined, all in vitro binding experiments have yielded an at least 1000-fold greater

affinity of the UBA domains for tetra-Ub compared to mono-Ub (22,54). Therefore it is very

likely that in the presence of polyubiquitinated substrates, the main binding partner of UBA

domains might be these poly-Ub chains rather than monoubiquitinated substrates or free Ub

unless the concentration of polyubiquitinated substrates is very low. The molecular nature of the

tighter binding of UBA to poly-Ub is not yet clear; however, structure analysis (58) as well as

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mutagenesis data on poly-Ub (59) suggest that they do not form plain linear poly-protein chains

like pearls on a string, but rather adopt globular structures. Hence additional contacts between

the linked Ub moieties and possibly other epitope(s) of the UBA domain might lead to the

increase in affinity in comparison to monomeric Ub.

Since UBA domains are often associated together with Ub-like domains in modular

proteins, the UBA domains could act as poly-Ub “receptors” whereas the Ubl domain might

interact directly with the proteasome. However such a shuttle mechanism has not yet been

confirmed in vivo. The differences for UBA and CUE domains in their biological function as

well as in the probable binding target, monomeric Ub versus poly-Ub, make it plausible that the

binding mechanisms of CUE and UBA domains do not need to be identical.

Interestingly, our de novo model was recently at least partially confirmed by results by

Walters and co-workers (41). Based on chemical shift differences between the isolated Ubl and

UBA domains and the domains in the context of the full length HHR23A protein, it was

concluded that in full-length HHR23A the Ubl domain interacts in a dynamic fashion with the

individual UBA domains and this interaction has a 1:1 stoichiometry, i.e. Ubl is exchanging

between one or the other UBA domains. No interdomain NOEs were observed, consistent with

very weak binding. Using residual dipolar couplings measurements, Walters and coworkers tried

to define the relative orientation of the domains in the modular protein (41). Although the

coordinates for their models of the interaction between Ubl and the UBA domains are not

available, using the figures of their publication we find a striking similarity between their models

for Ubl-UBA interaction and our Ub-UBA de novo models. Similar to the interaction with Ubl,

an identical surface epitope of Ub is involved in the binding to both UBA(1) and UBA(2)

domains. Helix 1 of either UBA(1) (residues 191-199) or UBA(2) (residues 348-356) contacts

the five-stranded β-sheet of either Ub or Ubl domain. For UBA(1) the relative orientation of the

helix in the model is the same as observed for HHR23A Ubl-UBA(1). For UBA(2), the

orientation of helix 1 is rotated by 180° in our de novo model compared to the results of Walters

et al. (41). In contrast to our chemical shift mapping studies and those performed by Ryu et al. on

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HHR23B UBA domains (38), Walters and co-workers (41) propose that only one helix of the

UBA domains contributes to the interaction with the Ubl domain. We find that the binding of the

UBA domains to Ub includes residues from both helices 1 and 3. This difference might explain

the much lower affinity of UBA domains for Ubl (KD ~2 mM) (38) than for Ub (KD ~300 to

500 µM). In summary, these results indicate that the interaction between Ub and UBA domains

might be different from the binding to Ub found for the structurally homologous CUE domain in

solution. However, further experimental data is required to understand how UBA domains

interact with Ub on a molecular level and whether the binding mechanism is different from the

CUE domains.

Finally, we note that RAD23 has been described as a binding partner through its UBA(2)

domain for various proteins involved in DNA repair and cell cycle control, e.g. HIV-1 Vpr (25),

methyladenine DNA glycosylase (MPG) (26), p300/cyclic AMP responsive element binding

(CREB)-protein (27), and peptide:N-glycanase (Png1) (28). Since the UBA-binding epitopes of

these proteins exhibit very different structures, the UBA domain of RAD23 must be able to bind

to various structural architectures. Therefore the binding mechanism of UBA domains is also

likely to vary for the diverse interacting proteins.

Acknowledgements

We thank Dr. Alexander Varshavsky for the gift of yeast ubiquitin expression plasmid

pET11c-Ub, Dr. Carina Johansson, Darian Cash and Nathan Cho for performing some of the

NMR experiments, and Evan Feinstein for manuscript and figure preparation. This work was

supported by NIH grant AI43190 to I.S.Y. Chen and J.F. The coordinates for the complex

models are available on request.

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Biochemistry 37, 2925-2934.

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Figure Captions

Figure 1: Chemical shift mapping of UBA(1) and UBA(2). (A and B) 500 MHz 1H-15N 2D

HSQC spectra of UBA(1) and UBA(2) free and bound to Ub. (A) 13C-,15N-labeled UBA(1)

free (black contour levels) and with 1:10 Ub (red) and (B) 15N-labeled UBA(2) free (black) and

with 1:10 Ub (red) at 27 ºC. Blue contours are used for side chain amide protons. (C and D)

Chemical shift change vs. sequence of UBA(1) and UBA(2). Graph representing the average

chemical shift change of the amide proton and nitrogen ∆δave of (C) UBA(1) and (D) UBA(2)

upon addition of 10 equivalent Ub. The dashed line indicate the threshold chosen for the color

coding used in Figure 1 E and F. Chemical shift changes of ∆δave smaller than 0.2 p.p.m. were

considered insignificant. (E and F) Binding interface of Ub on UBA(1) and UBA(2). Residues of

(E) UBA(1) and (F) UBA(2) shifting by more than 0.05 p.p.m. upon addition of Ub are marked

in orange. A representation of the molecular surface is shown on the left and a ribbon sketch is

shown on the right.

Figure 2: Chemical shift mapping of Ub. (A and B) 1H-15N HSQC of free Ub and Ub bound to

UBA(1) and UBA(2). 13C-,15N-labeled Ub in free conformation (black) and in complex with

(A) UBA(1) and (B) UBA(2) (red contour levels, ratio 1:4). Blue contour levels mark side chain

amide resonances. (C and D) Chemical shift change vs. sequence of Ub bound to UBA(1) and

UBA(2). The average chemical shift change (combined amide proton and amide nitrogen

chemical shift as for Figure 1 C and D) is shown for Ub bound to (C) UBA(1) and (D) UBA(2).

The dashed line indicates the threshold of chemical shift change used for Figures 2 E and F. (E

and F) Binding area of UBA(1) and UBA(2) on Ub. The changes of amide proton and nitrogen

chemical shift are displayed on the surface (left) and on a ribbon diagram (right) of human Ub as

classified in Figure 2 C, D. Residues are marked in orange according to the chemical shift

change (∆δave) on Ub upon addition of (E) UBA(1) and (F) UBA(2). For Ub binding to UBA(1)

(E) large changes (> 0.1 ppm.) for the chemical shifts are found for the residues L8, K11 (β1 -

β2 loop), I44 (β3), G47, K48, Q49 (β4), H68, L69 (β5), R72 and L73 (C-terminus). Residues

exhibiting smaller changes are V5, T7, G10, T12 (β1-β2 loop), I13 (β2), R42, L43 (β3), F45,

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A46 (β4), L50, Q62 (β4-β5 loop), L67 (β5) and L71 (C-terminus). For Ub binding to UBA(2),

residues L8, T9, G10, K11 (β1-β2 loop), I13, T14 (β2), R42, I44 (β3), G47, K48, Q49 (β4),

G53 (β4-β5 loop), H68, L69, V70 (β5), L71, R72 and L73 (C-terminus) of Ub show significant

chemical shift changes.

Figure 3: Differential chemical shift mapping of the Ub-UBA interaction. (A) Analysis of the

binding curve determined from chemical shift mapping of the Ub-UBA(1) interaction. The

average chemical shift change of the residue G174 of UBA(1) upon binding to Ub is plotted

against the concentration of Ub or Ub mutant. (B) As in (A) but for the interaction of Ub and

UBA(2). The average chemical shift change of residue G331 was used for analysis. (C)

Comparison of the chemical shift changes upon addition of Ub/Ub L8A to HHR23A UBA(1).

The boxes mark the residues for which a comparison of the chemical shift change of the Ub (red)

and Ub L8A (green) titration yielded a difference of more than 3-fold. The according residues

are annotated. (D) As in figure (C) but comparing the titration of Ub (red) and Ub R42A (green).

Figure 4: Homology models for the UBA(1)-Ub and UBA(2)-Ub interaction. (A) Model for the

interaction of HHR23A UBA(1) and Ub based on the structure of the complex of Cue2 CUE

domain and Ub, in (B) for the interaction of UBA(2) and Ub. Left panel: Ribbon sketch with the

structure of UBA(1) (A) and UBA(2) (B) displayed by a color ramp blue to red (N-terminus to

C-terminus). The helical axes of the UBA domains are displayed by dashed black lines, as a

reference for the orientation the axis of β-strand 5 of Ub is shown as dashed red line. Middle

panel: Interface between UBA(1) (A) or UBA(2) (B) and Ub, the surface of Ub is shown in atom

colors. Residues of UBA(1) or UBA(2) in direct contact with Ub are shown and labeled. Right

panel: As for middle panel but residues of Ub (grey) in direct contact with residues of UBA(1)

(A) or UBA(2) (B) (dark grey) are shown and labeled.

Figure 5: De novo docking models for the UBA(1)-Ub and UBA(2)-Ub interaction. Similar to

Fig. 4, the interaction of (A) UBA(1) and (B) UBA(2) with Ub is shown. The complexes were

obtained by computational docking using ambiguous restraints based on the chemical shift

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mapping results. Left panel: The helical axes of the UBA domains (UBA(1): in A; UBA(2) in B)

are represented by dashed black lines, the orientation of the UBA domain in respect to Ub can be

determined from the axis of β-strand 5 of Ub shown as red dashed line. Middle panel: The

interface between UBA(1) (A) and UBA(2) (B) and Ub. The surface of Ub is shown in atom

color; the residues of either UBA domain in contact with Ub are shown and labeled. Right panel:

As for middle panel, but residues of Ub in direct contact with UBA(1) (A) and UBA(2) (B) are

shown in grey and labeled.

Figure 6: Sequence alignment of UBA and CUE domain proteins. The sequences of several UBA

domains (SWISS-PROT entry codes are indicated) and CUE domains were aligned based on a

structural superposition of HHR23A UBA(1) and Cue2 CUE domain. The helices are marked by

boxes, the arrows indicate residues which either exhibit changes of their chemical shift upon

binding to Ub (upper panel, UBA domain) or which are buried in the complex (lower panel,

Cue2 CUE-Ub). The amino acids are color coded using green for hydrophobic residues

(A,F,I,M,L,V,Y,W), red for negatively, blue for positively charged residues (H,K,R), and orange

for polar amino acids (N,Q,S,T). The consensus sequence for UBA domains was taken from the

SMART database (http://www.embl-heidelberg.de/smart), the symbols for the amino acid

grouping are as follows: l = aliphatic (I, L, V); . = any; a = aromatic; c = charged; h =

hydrophobic (A, C, F, G, H, I, K, L, M, R, T, V, W, Y); p = polar; + = positively charged; - =

negatively charged; s = small (A, C, D, G, N, P, S, T, V); u = tiny (A, G, S) and t = turnlike (A,

C, D, E, G, H, K, N, Q, R, S, T).

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Thomas D. Mueller, Mariusz Kamionka and Juli FeigonSpecificity of the interaction between UBA domains and ubiquitin

published online January 5, 2004J. Biol. Chem.

10.1074/jbc.M312865200Access the most updated version of this article at doi:

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specificity of the interaction between uba domains and ubiquitin · 2004-01-05 · minimization and...

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