Biochemical and Biophysical Research Communications 290, 1214–1219 (2002)

doi:10.1006/bbrc.2001.6267, available online at http://www.idealibrary.com on

Identification of Structural Motifs and Amino Acidswithin the Structure of Human Heparan Sulfate 3-O-Sulfotransferase That Mediate Enzymatic Function

Rahul Raman,* James Myette,* Ganesh Venkataraman,† V. Sasisekharan,†and Ram Sasisekharan*,1

*Division of Bioengineering and Environmental Health and †Harvard–Massachusetts Institute of TechnologyDivision of Health Sciences and Technology, Massachusetts Institute of Technology,77 Massachusetts Avenue, Cambridge, Massachusetts 02139

Received November 26, 2001

intracellular signaling, cell–cell interactions, and both

In an accompanying paper [J. R. Myette, Z. Shriver,

J. Liu, G. Venkataraman, and R. Sasisekharan (2002)Biochem. Biophys. Res. Commun. 290, 1206–1213], wedescribed the purification and biochemical character-ization of a soluble, recombinantly expressed form ofthe human heparan sulfate 3-O-sulfotransferase (3-OST-1). Such an important first step enables detailedstructure–function studies for this class of enzymes.Herein, we describe a complimentary, structure-basedhomology modeling approach for predicting 3-OST-1structure. This approach employs a variety of struc-tural analysis and molecular modeling tools used inconjunction with protein crystallographic studies ofrelated enzymes. In this manner, we describe impor-tant motifs within the predicted three-dimensionalstructure of the enzyme and identify specific aminoacids that are likely important for enzymatic func-tion. © 2002 Elsevier Science (USA)

Heparin-sulfate glycosaminoglycans (HSGAGs) arepresent at the cell surface and throughout the extra-cellular matrix as the complex linear polysaccharidecomponents of proteoglycans. This particular class ofglycosaminoglycans, once commonly believed to serveexclusively as structural components of the extracellu-lar milieu, is now known to play a dynamic functionalrole in a diverse number of biological events related to

Abbreviations used: HSGAG, heparan sulfate glycosaminoglycan;3-OST-1, recombinant human heparan sulfate 3-O-sulfotransfer-ase-1; NDST-1, heparan sulfate N-deacetylase-N-sulfotransferase-1;PAPS, 39 phosphoadenosine-59 phosphosulfate; NMP kinases, nucleo-side monophosphate kinases; SLH, b-strand–loop–a-helix; PSB;phosphosulfate binding loop, PB; phosphate binding loop.

1 To whom correspondence and reprint requests should be ad-dressed at MIT, 77 Massachusetts Ave., Building 16-561, Cam-bridge, MA 02139. Fax: 617-258-9409. E-mail: rams@mit.edu. http://web.mit.edu/tox/sasisekharan/.

12140006-291X/02 $35.00© 2002 Elsevier Science (USA)All rights reserved.

cell and tissue morphogenesis (1). This diversity andspecificity of HSGAG function follow from the exten-sive chemical heterogeneity and tissue-specific expres-sion pattern of these biopolymers. This structure-function relationship is ultimately dictated, in turn, bya regulated and likely concerted HSGAG biosyntheticprogram that involves a number of functionally relatedenzymes.

The biosynthesis of heparin/heparan sulfate (2) es-sentially begins with the linear assembly of the disac-charide repeat unit (or so-called HSGAG “buildingblock”) comprised of uronic acid (a-L-iduronic or b-D-glucuronic) linked 134 to a-D-glucosamine (3–6). Theuronic acid–glucosamine backbone is then variablymodified at structurally specific positions that includeacetylation or sulfation at the N-position of the glu-cosamine, epimerization of glucuronic acid to iduronicacid (7, 8) and additional sulfations at the 2-O positionof the uronic acid (9) and/or the 3-O, 6-O position of theglucosamine (10–14), each occurring nonrandomly atvarious positions within the polysaccharide backbone.It is the specific sulfation pattern for each HSGAGchain that imparts a distinctive structural “signature”to each polysaccharide. This signature correspondinglydictates a specific HSGAG-protein interaction that isintegral to a particular HSGAG-modulated biology.

A quintessential example of such a HSGAG signa-ture is that of the highly specific 3-O-sulfation of glu-cosamine occurring in select structural domains withinan oligosaccharide chain. To date, five isoforms of 3-O-sulfotransferase have been identified (12). Evidenceexists that expression of these isoforms is highly reg-ulated in a tissue and temporal manner. For such areason, the activity of this particular HSGAG sulfo-transferase may be of special biological importancebeyond its role in coagulation or thrombosis. A detailed

understanding of the structural basis for the 3-O- were superimposed on that of the PAP cofactor. Interestingly, this

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sulfotransferase catalytic mechanism and substratespecificity of the respective isoforms would greatly ad-vance our understanding of the mechanisms involvedin the synthesis of specific HSGAG sequences impor-tant for biological activity. In turn, the biochemicalcharacterization of this and other heparan sulfate sul-fotransferases would be greatly facilitated by the facileexpression and purification of large amounts of activeenzyme. For 3-OST-1, effective recombinant expressionin Escherichia coli and the subsequent purification ofhighly active enzyme has been achieved (24). What islacking is the availability of a detailed structuralframework to orient additional biochemical studies.

Toward this latter end, we have investigated themolecular features of human 3-OST-1 using a system-atic homology-based modeling approach. This paperdescribes such a structural framework that is largelybased on currently available cocrystal structures ofseveral different sulfotransferases, each one contain-ing the sulfate donor analog 39 phosphoadenosine 59phosphate (PAP). The PAP binding region is a highlyconserved structural motif in all the sulfotransferasesexamined (15, 16). As this PAP binding domain is alsostructurally very similar to the nucleoside phosphatebinding motifs present in nucleoside monophosphate(NMP) kinases (15, 17) we used known NMP kinaseco-crystal structures as a refinement to our modeling,in particular to identify candidate PAP binding resi-dues and to provide further structural constraints onthe PAP binding site of 3-OST-1. The putative sub-strate-binding region of 3-OST-1 was modeled based onthe recently solved sulfotransferase domain (NST) ofthe human N-deacetylase–N-sulfotransferase-1 (NDST-1)(18).

MATERIALS AND METHODS

The sequence of human heparan sulfate 3OST-1 was obtainedfrom GenBank database (pid: g4826764). The co-crystal structures ofthe sulfotransferases—pdb ids: 1NST (NST), 1AQU (mouse estrogensulfotransferase; mEST), 1CJM (catecholamine sulfotransferase),1EFH (hydroxysteroid sulfotransferase) and the NMP kinases (pdbids: 1UKZ, 1UKD, 1ECK, 1AKY, 1GKY, 1TMK, 1VTK) were ob-tained from the protein data bank (www.pdb.org).

The PAP binding site is comprised of a 59 phosphosulfate binding(PSB) and a 39 phosphate binding (PB) loop, and a third loop thatinteracts with the adenine ring (SLH3), each of which is flanked bya b-strand and an a-helix forming a strand–loop–helix structure.The common structural features in the cofactor binding region of theNMP kinases and the sulfotransferases were the 59 PSB loop (59PBloop in the kinases) and the SLH3 loop which interacted with theadenine ring (common to both PAP and ADP). A first pass compar-ison of the PAP binding site in all the sulfotransferases was per-formed by superimposing the 59 phosphate backbone (59P-O-C5-C4)of the PAP cofactor in all the crystal structures. This superpositionaligned the three structural strand–loop–helix binding motifs. Thestructural comparison was further refined by superimposing the Caatoms of the aligned binding motifs. Similarly the 59 phosphatebackbones of the ADP cofactor in the NMP kinase crystal structures

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superposition aligned the PB loop and the SLH3 loop of the kinaseswith those of the sulfotransferases indicating that these three motifswere structurally conserved in all these enzymes. The structuralcomparison was further refined by superimposing the Ca atoms ofthe aligned binding motifs in the kinases and sulfotransferases.

The putative PAPS binding residues on 3-OST-1 were identifiedbased on their propensity to thread into the conserved structuralmotifs in the NMP kinases and sulfotransferases. Since a wealth ofstructural information was available, these residues were assignedwith a good degree of confidence. The best template for identificationof putative substrate binding residues was the cocrystal structure ofNST with PAP (pdb id: 1NST) since the broad definition of substratefor both these enzymes is heparan sulfate. The coordinates of theresidues involved in PAP binding and substrate binding were as-signed based on the crystal structure of NST, where the alignment ofthe PAP binding site was obtained based on the structural propen-sity of the putative residues and the substrate binding was obtainedby sequence alignment. The position of the PAP cofactor relative to3-OST-1 was fixed using the NST co-crystal structure. Random loopswere generated in 3-OST-1 corresponding to the regions 587–600,665–669, and 880–882 that were disordered in the NST crystalstructure. Assignment of coordinates and generation of loops wereperformed using the homology modeling module of INSIGHTII. Thedeletions in the modeled 3-OST-1 structure were closed by perform-ing 100 steps of Newton–Raphson minimization (with chargesturned off) by fixing the coordinates of most of the protein and justallowing the region around the deletion to move freely. The entiremodeled structure was subjected to 150 steps of steepest descentminimization without considering charge effects followed by conju-gate-gradient minimization including charge effects until the rmsderivative of energy was ,0.01 kcal/mol. A. All the minimizationruns were carried out using the discover module of INSIGHTII.

RESULTS AND DISCUSSION

From our analysis of the crystal structures of thesulfotransferases, we observed that the cofactor-bind-ing region was distinct from the substrate binding re-gion of the enzyme indicating a common theme in thesulfuryl transfer mechanism by these enzymes. Al-though these enzymes had little sequence homologyand drastically different folds, their cofactor (PAP)binding region was structurally very similar. However,the topology of the substrate-binding site appeared todepend on the type of substrate and also governed theactivity of the enzyme. Based on our observations, wedecided to dissect the 3-OST-1 structure into respectivecofactor (PAPS) binding, substrate binding, and cata-lytic regions and look at each of these regions in turn.

PAP Binding

The PAP binding site of 3-OST-1 was modeled with ahigh degree of confidence since this structural motif ishighly conserved in all the sulfotransferases andshares a high structural homology with the NMP ki-nases (Fig. 1). The PAP binding site in our modeled3-OST-1 is topologically similar to the correspondingregions of other sulfotransferases consisting of the 59PSB, 39 PB and the SLH3 motif (Fig. 2A).

Lysine (K64), glycine (G66), and threonine (T67) res-idues in the putative 59 PSB loop are highly conserved

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in the sulfotransferase family (Fig. 1). The backboneatoms of K64 and G66 and the Og atom of T67 appearto be in close proximity to oxygen atoms of the 59phosphate group of PAP (Fig. 2B). The residues equiv-alent to K64 in NST (K614) and K48 in estrogen sul-fotransferase (K48) have been shown to be critical foractivity (19, 20). Another residue that appears to beclose to the 59 phosphate oxygen atom is K270. Al-though this residue is not highly conserved in everymember of the sulfotransferase family it is conservedamong the different isoforms of NDST and 3-OST. Thisresidue appears to be positioned in the correct orienta-tion to stabilize the leaving 59 sulfate group of PAPS.Other residues in the 59 PSB loop that could potentiallyinteract with the phosphate group are R68 and A69. Inaddition to its interacting with the 59 phosphate group,R68 also has its side chain in the right orientation tocontribute to heparin binding.

G271, R272, and H274 residues in the putative 39 PBloop are positioned to interact with the 39 phosphategroup of PAPS. In addition, the Nh of R147 and the Ogof S155 appear to be in close contact with the oxygenatoms of the 39 phosphate group. S155 is also a highlyconserved residue in the sulfotransferase family. Thecorresponding serine residue (S712) in NDST-1 existsin the same spatial position as S155 in 3-OST-1 andinteracts with the 39 phosphate group as demonstratedin the crystal structure of NDST-1 (18).

In addition to the 59 and 39 phosphate binding loops,a hydrophobic pocket comprising of F254, I221, A69and L220 and L266 appears to stack the adenine ringof PAPS, thereby fixing the orientation of PAPS rela-tive to the enzyme. Of these residues, the phenyl ringof F254 is parallel to the adenine ring (Fig. 2B). Thisphenylalanine serves a similar stacking function to

FIG. 1. Comparison of cofactor binding sites in sulfotransferb-strand–loop–a-helix motifs constituting the cofactor (ATP/PAP) biferases is shown. The completely conserved residues are shown in bolcorresponding pdb ids. The residues constituting the PAP binding susing the structural alignment of the NMP kinases and the sulfotra

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F816 in the NST structure. Residues L220 and I221are a part of the SLH3 motif.

Substrate Binding

The substrate binding site of 3-OST-1 was modeledusing the NST crystal structure as a template (Fig. 3).We find that this binding site is an open cleft withabundant basic residues at the base of the cleft that arearranged in such a manner as to provide strong ioniccontacts with the sulfate groups of the heparin chain.The size of the cleft appears to accommodate at least anHSGAG tetrasaccharide. The basic residues R193,R63, R68, and R264 line up along the base of the cleftwith the side chains and point toward the open end(Fig. 4). The outer edge of the cleft also possesses somebasic residues that include K119, N85 and Q166. Theseresidues may provide additional stabilizing contactsfor the binding of the HSGAG substrate. Recently,isoform-specific 3-O-sulfotransferase domains havebeen proposed from domain swapping experiments(21). In this study, substrate specificity for each of therespective sulfotransferases is attributed to noncon-served regions within the putative C-terminal sulfo-transferase domain that flank the substrate bindingcleft as inferred from a comparison of the primarysequences of several NDST and 3-OST isoforms. Manyof these amino acids, in fact, map to the correspondingregion of the heparin binding pocket in our 3-OST-1model.

Cystine Loop

Most of the heparan sulfate sulfotransferases con-tain a loop region that is flanked by two cysteine resi-dues. This so-called cystine loop is present in close

s and NMP kinases. Structure-based sequence alignment of theng site in the crystal structures of NMP kinases and the sulfotrans-ce and highlighted in gray. The enzymes are abbreviated using theirof 3-OST-1 (marked as 3OST and numbered below) were assignederases as a guiding framework.

asendid faitensf

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proximity to the hydrophobic pocket stacking the ade-nine ring in both the NDST-1 (between C818 and C828)and 3-OST-1 (between C255 and C266) structures (Fig.2A). In both structures, the two cysteines are spatiallyproximal to one another and presumably form a disul-fide bond (18). While the exact functional role of thisloop has not been determined, the location of this looprelative to the heparin binding cleft suggests a possiblerole of this region in modulating substrate bindingand/or specificity. If, in fact, this is the case, then onecould envision that subtle differences in either looplength or regional amino acid composition may influ-ence this structure–function relationship.

Putative Catalytic Residues

Basic residues K64, R68, H164, K119, and K270,R273 surround a region in the 3OST-1 that is close to

FIG. 2. PAP Binding site in 3-OST-1. (A) Stereo view of the predtrace colored in green), 39 PB loop (ribbon trace colored in purple) andin red. The residues interacting with the 59 and 39 phosphate groupsto the PAP binding site is shown as a ribbon trace colored in dark brPAP cofactor.

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the 59 phosphate group of PAP. From their proximity tothe 59 phosphate group, it appears that some of theseresidues may be potentially involved in catalysis. Asimilar catalytic site has been observed for mouse es-trogen sulfotransferase, which involves a lysine and ahistidine (17). In addition, several of the substratebinding residues in 3-OST-1 share homologous resi-dues in the NDST-1 crystal structure, where they havebeen implicated in playing a critical role both in bind-ing heparan sulfate and catalysis (20, 22). These resi-dues in 3-OST-1 include K64, R68, H164, K119, R264,and R273.

In lieu of an available high-resolution X-ray crystalstructure for the human heparan sulfate 3-O-sulfo-transferase-1, we have taken a systematic homology-based approach to probe the structural features of thisenzyme. In this strategy, we first used known crystal

d PAP binding site in 3-OST-1 comprised of the 59 PSB loop (ribbonSLH3 loop (ribbon trace shown in blue). The PAP cofactor is colored

e colored in dark blue. The cysteine loop which is in close proximity. (B) 3-OST-1 active site detailing the residues interacting with the

ictethear

own

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structures of different classes of sulfotransferases (in-cluding the HSGAG sulfating enzyme NDST-1) in con-junction with the NMP kinase family of enzymes toidentify the putative PAPS binding site in 3-OST-1.Given the structural conservation of the nucleoside-phosphate cofactor binding site throughout these twoenzyme families, this homology-based modeling strat-egy is both logical and highly informative. To identifycandidate HSGAG binding residues, we focused ourstructural analysis on a direct comparison between3-OST-1 and the HSGAG sulfotransferase NDST-1.

FIG. 3. ClustalW alignment of 3OST-1 and NST. ClustalW alsequence of NST extracted form the crystal structure (pdb id: 1NST)Residues in bold face that are also shaded in gray constitute the pu

FIG. 4. Structure of 3-OST-1 and putative heparin binding cleft.CPK rendered structure of the modeled 3-OST-1 (gray) with theputative heparin binding site colored in black. A stick representationof a model heparin tetrasaccharide [constructed using NMR data(23)] is shown to illustrate the likely positioning of the substrate inthe putative heparin binding cleft.

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While these two enzymes do not share extensive se-quence similarity, it appears, nevertheless, that mostof the critical substrate binding residues are conservedbetween the two functionally-related sulfotransfer-ases. It remains to be experimentally determined, how-ever, how these residues may specifically interact withthe HSGAG chain and/or play a role in catalysis. Takentogether, the structural information suggests thatthere is a common biochemical mechanism of sulfuryltransfer shared by these sulfotransferases.

Given the functional importance of HSGAGs from abiological perspective, the in vivo activities of 3-OSTand other HSGAG modifying enzymes are highly rele-vant. Possessing a detailed understanding of the un-derlying molecular basis for these activities is thusabsolutely critical. In the previous paper, we have dem-onstrated how one HSGAG sulfotransferase in partic-ular, 3-OST-1 can be recombinantly expressed in E.coli and subsequently purified in such a manner toyield milligram quantities of soluble, highly active en-zyme for both biochemical and structural studies (24).In conjunction with this in vitro work, we now providean informative structural framework to orient futurebiochemical investigations. Most importantly, perhaps,our results give validation for expanding this tandemapproach to a study of additional heparin/heparan sul-fate sulfotransferases as a first-step toward elucidatingimportant structure–function relationships.

ACKNOWLEDGMENTS

This work was supported in part by funding from NIH GrantsGM057073 and HL059966 to R.S. R.R. is a recipient of the Merck–MIT fellowship and J.M. is a recipient of the Toxicology TrainingGrant, MIT (5T32GM08334).

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