Molecular Modeling of the Amino-Terminal Zinc Ring Domain of BRCA1

CANCER RESEARCH56. 2539-2545. June I, 19961 Molecular Modeling of the Amino-Terminal Zinc Ring Domain of BRCA1

Rachelle J. Bienstock,1 Tom Darden, Roger Wiseman, Lee Pedersen, and J. Carl Barrett

National institute of Environmental Health Sciences, Laboratories of Quantitative and Computational Biology fR. J. B.. T. D., L P.] and Molecular Carcinogenesis IR. W.. J. C. B.J, Research Triangle Park. North Carolina 27709

ABSTRACT in Fig. 1. Although a common functionality for the zinc ring domain has not been identified, it appears to interact with DNA either through The equine herpes virus zinc ring domain nuclear magnetic resonance direct binding or indirectly by mediating protein-protein interactions. structure was used for homology-based modeling of the amino-terminal The zinc ring domain of several proteins has been shown to exhibit zinc ring domain of the BRCA1 breast and ovarian cancer susceptibility gene. The zinc ring domain of BRCAJ is of particular interest because it DNA-binding activity (10) or nuclear localization (11â€”13). is the location of significant and frequently occurring missense (Cys6tGly, The BRCA1 zinc ring domain and its structure and function are of Cysâ€•Gly,andCysâ€•Tyr)andframeshift (lSSdelAG) mutations observed particular interest, because it is the location of some of the most In several high-risk kindreds. The BRCA1 zinc ring domain possesses frequently occurring mutations linked to breast and ovarian cancer. 54% sequence similarity with the equine herpes virus zinc ring domain. The l85delAG mutation (deletion oftwo nucleotide base pairs in exon The model structure undergoes little conformational variance after 140 ps 2) in the zinc ring domain has been shown to occur in 1 in 100 of solvated molecular dynamics. This model proposes BRCA1 zinc ring Ashkenazi Jewish individuals (14, 15). This is in comparison with the domain residues that may play a role in DNA binding and/or protein estimated I in 833 frequency of all BRCAI mutations in the general protein interactions. These predictions provide a point of departure for population (14, 15). In this article, a three-dimensional model for the the design of mutants to probe BRCA1 zinc ring domain functionality. zinc ring domain of BRCA1 is presented. This model proposes po tential BRCAI zinc ring domain residues that may play a role in the INTRODUCTION function of this domain, either binding directly to DNA or mediating DNA binding through protein-protein interactions. BRCAJ, the breast and ovarian cancer susceptibility gene on human chromosome l7q21, was recently identified (1, 2). Mutations in this gene account for approximately 95% of families with increased mci MATERIALS AND METHODS dence of early onset breast and ovarian cancer and 45% of families with increased incidence of breast cancer alone (3, 4). BRCAJ is Sequence Alignment. The three-dimensional structures of two zinc ring believed to encode a tumor suppressor protein, because most muta domains, the EHV2and the acute PML proto-oncoprotein, have recently been tions appear to result in loss of function. This gene is expressed in determined by multidimensional NMR (8, 9, 1 1). The sequence alignment several tissues, including breast and ovary, and encodes a I863-amino between the PML and EHV zinc ring domains is shown in Fig. 2 along with the secondary structural elements identified for each protein from the solved acid protein. NMR structures. Several criteria were used to select the EHV ring domain The amino-terminal domain of BRCA1 possesses high sequence structure rather than the PML structure as the basis for the BRCA1 model. The homology with a zinc finger topology (1). This zinc finger topology, BRCAI zinc ring domain, for example, possesses greater sequence similarity C3HC4,isreferredtoastheâ€œzinc-ringdomain,â€•â€œRINGfinger,â€•or to EHV than to PML. Using a sequence alignment of Smith and Waterman (16) A-Box (5) and is found in several viral proteins, proto-oncoproteins, with standard default parameters, the BRCA I zinc ring domain possesses 54% and regulatory and transcription factors (6). These include develop sequence similarity and 38% sequence identity with the EHV zinc ring domain, mental regulatory proteins [XNF-7, SINA, PSC, SU(z)2, and DG17}, compared with 42.8% sequence similarity and 30.6% sequence identity to the cell regulatory proteins (lAP and DROC3H), signal transduction PMLdomain.Fig. 2 shows the sequencealignmentbetween the BRCA1 and proteins (CRAF1), human oncoproteins (MDM2, MEL18, BMI-l, EHV and PML ring domains,respectively. RET, T18, CBL, and PML), proteins that regulate gene expression A BLASTP (Basic Local Alignment Search Tool for Proteins; Ref. 17) search of the Brookhaven Data Bank selected and aligned the EHV with the (RPT-l, PAS4, TLR, and HIP116), viral proteins, including some BRCA1 zinc ring domain, and yielded a P of 9.6 X l0@ (chance likelihood viral gene expression proteins, (lEl 10, VZ61, CG3O, PE-38, EPO, of the two sequences relationship). Folding search programs, such as Pro EHV, LCMV, and P11), and proteins involved in DNA repair or files-3D by Luthy et a!. (18), also indicated that the EHV and BRCA1 zinc ring recombination (RAD5, RADI 8, RAD16, RAG- 1, and UVS2). protein domains have similar folds. Hydrophilicity plots of Kyte and Doolittle The zinc ring family, first identified in 1991 (7), was found to (19), surface probability [calculated according to the method of Emini et a!. possess two finger-like domains connected by a linking region and to (20)], chain flexibility predictions (Karplus-Schulz), secondary structure pre require zinc for folding. The zinc ring C3HC4 sequence folds inde dictions of turns and a helices by Chou and Fasman (21), turns prediction by pendently and is a distinct stable domain (8, 9). This domain differs Gamier et al. (22), and the antigenicity index of Jameson and Wolf (23) all structurally from other zinc finger domains in that the first and third support greater similarity of the EHV ring domain structure to BRCAI than to pairs of cysteines form one zinc-binding site, and the second and PML(datanot shown). A multiple-sequencealignmentof the threesequences (the Pileup algorithm from the package below; Refs. 24 and 25) yields pairwise fourth pairs of cysteines form the second metal-binding site. This similarity scores ofO.44 for BRCA1 and EHV, 0.37 for BRCAI and PML, and crossed pattern is similar in configuration to a 1â€”3, 2â€”4 disulfide 0.42 for EHV and PML. Therefore, EHV was used as the basis for developing bond pattern. A schematic of the BRCA1 amino-terminal zinc ring a model for the BRCA1 zinc ring domain. domain with cysteine and histidines ligated to zinc indicated is shown Several multiple sequence alignments have been published previously, aligning the BRCAI zinc ring domain sequence to the sequences of other Received I2/28/95; accepted 4/3/96. known ring domains. The alignment by Borden et a!. (11) also shows greater The costs of publication of this article were defrayed in part by the payment of page sequence similarity between BRCA1 and EHV than between BRCA1 and charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I To whom requests for reprints should be addressed, at National Institute of Envi 2 The abbreviations used are: EHV, equine herpes virus; PML, promyelocytic leuke ronmental Health Sciences, P.O. Box 12233, Mail Drop 10-03, Research Triangle Park, mia; NMR, nuclear magnetic resonance; CVFF, consistent valence force field; kcal, NC 27709. Phone: (919) 541-3397; Fax: (919) 541-1578; E-mail: rachelle@ kilocalorie; RMS, root-mean-square; HSV, herpes simplex virus; CRD, cysteine rich picard.niehs.nih.gov. domain of protein kinate C; WT1,Wilms' tumor gene; TFIIIA, transcription factor III A. 2539

The two zinc ions were entered into the structure by superposition with the herpes virus structure, which gave a general positioning for the zinc ions in the protein. The resulting structure was energy minimized in vacuo using a distance-dependent dielectric constant, as described by Guenot and Kollman (28). The ligand-zinc ion distances were constrained in the minimization to the distances found between the corresponding ligands and zinc in the herpes virus protein. The zinc-cysteine sulfur atoms were pulled together using 500 cycles of conjugate gradients with a force of 100 kcal/mol, followed by minimization with 500 cycles of steepest descents and 1000 cycles of conjugate gradients until the maximum derivative was less than 0.1 kcal/A. All constraints were R then removed, and the structure was reminimized with 1000 cycles of conju F SQ L gate gradients until the maximum derivative was less than 0. 1 kcal/A. Charges were assigned to the zinc ions and cysteine ligands Sy and histidine Nbl . Because of the ionic and covalent nature of the zinc ligand interactions, the zinc ion charges are reduced and assigned values of + I.5 in the four cysteine-ligand environment and + 1.9 in the three-cysteine- and one-histidine ligand environment. All cysteine Sys have negative partial charges, because a hydrogen is abstracted on binding with zinc. The histidine N81 also has a negative partial charge on binding with zinc. In this model, the cysteine Sys in L the four-ligand case were assigned partial charges of â€”0.275,andcharges of I â€”0.450wereassigned in the three-ligand case. The histidine N bound to the MQK zinc was assigned a partial charge of â€”0.670. Distances were constrained in a several-step minimization (in vacuo) until

Fig. 1. Schematic of the BRCA1 zinc ring domain with cysteine and histidines ligated the ligand-zinc distance criteria were met. This procedure minimized energy to zinc indicated. strain and prevented bumping and overlap of atoms. The average zinc ligand distance in the constrained minimized structure is 2. 1 A. The structure was then reminimized with no constraints. The model was considered acceptable PML. The sequence alignment by Zhou et a!. (26) was not designed as a when reasonable zinc ligand distances could be maintained after removal of structural alignment and does not consider the EHV sequence in the alignment. constraints and reminimization. Pairwise sequence alignments were performed using the Genetics Computer Molecular Dynamics. Moleculardynamicssimulationswereperfonnedto Group Wisconsin Sequence Analysis package, version 8, Bestflt alignment test the stability of the zinc ring BRCA1 model structure. The structure algorithm of Smith and Waterman (16). The Pileup (24, 25) alignment algo obtained from constrained minimization was solvated in a s-A layer of water rithm was used for multiple sequence alignments. and subjected to 140 ps of molecular dynamics. No distances were constrained The EHV-l (Brookhaven Data Bank entry file, 1CHC) zinc ring domain during the molecular dynamics. The molecular dynamics simulation (using the was aligned with that of the BRCA1 protein as described in Fig. 2. Residues Biosym CVFF) was performed with nonperiodic boundary conditions using 1â€”67ofthe 1CHCprotein aligned with residues 18â€”86ofthe BRCAI protein. the cell multipole method (fourth-order multipole) for treatment of nonbonded Alignment of the cysteine and histidine residues that form the stabilizing Coulombic and van der Waals interactions. A distance-dependent dielectric ligand-zinc interactions is maintained in both proteins. Secondary structural constant was used (28). The structure was initially reminimized with solvent, elements in the EHV are shown above the sequence. The EHV possesses an and dynamics were performed beginning with the energy-minimized, solvated antiparallel 13sheet that begins at the amino terminus, followed by an a helix structure. The dynamics step size was I fs and structures, were saved along the and a final single (3sheet. Fig. 3 is a graphical sketch of the EHV, illustrating trajectory every 2 ps. Calculations were performed on a Silicon Graphics the secondary structural elements and their relationship to the zinc ions and Indigo2 (MIPS R4400; 150-MHz central processing unit) computer. ligands. Homology Modeling. A three-dimensional structural model was devel RESULTS o_ for the amino terminus (residues 18â€”86)ofBRCA1 constituting the zinc ring domain using homology modeling. The coordinates of the average NMR Structural differences have been identified between the PML and structure (mean average over 40 NMR structures) of the EHV (entry file, the EHV NMR-determined zinc ring structures (11). The PML struc 1CHC; Brookhaven Protein Data Bank) served as the basis structurefor ture is composed of a @3strandfollowed by two antiparallel @3strands, @ homology modeling. The coordinates of the aligned secondary structural one turn of a helix, and a fourth f3 strand. The EHV structure elements were applied to the BRCA1 sequence. The secondary structural consists of two antiparallel @3strands followed by an a helix and a elements proposed for BRCA1 correspond to fJ sheet 1 (residues 34â€”38),@3 third @3sheet. Although EHV and PML structural differences have sheet 2 (residues 41â€”45), a helix (residues 46â€”54),and f3 sheet 3 (residues been used to support the idea that the ring motif is structurally diverse, 71â€”76). There were two insertions in the BRCA1 structure, residues 58â€”60 and 69. Therefore, it was necessary to insert protein loops. Homology mod two-layer a/;3 proteins such as EHV and f3proteins such as PML have cling was performed using the Biosym Technologies homology module, ver been shown to have overall common folds (29). Both PML and EHV sion 2.3.5. BRCA1-inserted loops were modeled by beginning with the loop structures possess major conserved features: the â€œcross-braceâ€•Zn2@ search procedure of the homology modeling. Coordinates were suggested for binding arrangement; the topology of the central antiparallel @3 the protein loops, and the lowest energy loop structures were selected. The strands; and packing of the conserved residues in the hydrophobic structure developed was energy minimized after splicing the loops to resolve core (11). In both structures, the 6-nitrogen of the histidine is ligated discontinuities between the loops and a helix and @3sheet structures and to the zinc in place of the more common c-nitrogen. The C@RMS overlaps of side chains using steepest descents and conjugate gradient conver deviation between the two structures for residues comprising the first gence algorithms. zinc-binding site (Cys24, Cys27, Cys44, and Cys47) is 1.93 A, Calculations:EnergyMinimization.Calculationswereperformedusing indicating that this binding site is somewhat similar in both structures. the Biosym Technologies Discover version 94.0 program, using the CVFF modified to include nonbonded parameters for zinc. Only nonbonded zinc The majority of structural differences between PML and EHV occur interactions were considered. Parameters for the Lennard-Jones constant [po between the central (3strands and second zinc-binding sites, where the tential distance (Rij), 2.2 A; potential well (a), 0.0125 kcal/mol] were adapted amino acid sequences of the two ring-domains are dissimilar. from those used for carbonic anhydrase II (27). These values were used to Several subfamilies are beginning to arise within the zinc ring calculate the A and B constants required for the CVFF. protein family. Although the zinc ring occurs near the amino-terminus 2540

region of greatest structural dissimilarity

tâ€”@1l I@2â€”Ilahelixl Iâ€”f@3â€”I BRCA1 18 MQKILE ..CPICLELIKE .PVSTKCDHIFCKFCMLKLLNQKKGPSQCPLCKN ..DITKRSLQESTRFSQLVEEL 86 @ :1 111111 .:.:..IIIJ.l::::.l... liii EHV 1 MATVAE .RCPICLEDPSNYSMALPCLHAFCYVCITRWIRQNPT . . .CPLCKV . .PV .ESWHTIESDSEFGDQL 67 11.1.:: :..JlIII.:I.I:...I 11:1.: I: :: I PML 49 EEEFQFLRCQQCQAEAK CPKLLPCLHTLCSGCLEASGMQ CPICQAPWPL.GADTPALDNVFFESLQR 114 I-f311 1J321 1f331 â€”â€”â€”i I3i@I I@4I @ (helical turn) helix)

Fig. 2. Sequence alignment of EHV, PML, and BRCAI zinc ring domains illustrating secondary structural elements and the region of greatest structural dissimilarity. This alignment was used as the basis for developing a homology model for the BRCAI zinc ring domain. BRCA1 possess 54% sequence similarity and 38% sequence identity with the EHV zinc ring domain. BRCAI possessed less sequence similarity, 42.8%, and less sequence identity, 30.6%, with the PML zinc ring domain than with EHV, as shown in the alignment. Multiple sequence alignment of the BRCA1, EHV, and PML zinc ring domains yields pairwise similarity scores of 0.44 for BRCA1 and EHV, 0.37 for BRCAI and PML, and 0.42 for EHV and PML.

ring finger protein and the HSV type 1 ring domain are not function ally interchangeable. (HSV is required for activation of viral gene expression.) Neither a chimeric HSV protein that contained the PML ring finger nor PML was able to activate gene expression in assays (31). This work suggests that ring finger domains do not possess a common functionality. Different subcategories of zinc ring proteins may reflect structural classes of zinc ring domains with differing functionality. The final minimized BRCA1 zinc ring domain model is shown in Fig. 4 with cysteine and histidine zinc ligands. Superimposing the two minimized BRCA1 structures before and after removing the distance constraints, the RMS deviation is 0.02 A (all atoms), and the backbone

D67

@o6o

Fig. 3. Graphical sketch of the EHV illustrating secondary structural elements from the solved NMR structure. The zinc ring domain is a two-layer a/(3 protein. which from the @ NH2 terminus possesses an antiparallel sheet followed by an a helix, a large loop, and @ a final single sheet.

in most protein family members, there are several proteins for which the zinc ring occurs near the middle of the sequence (SINA, PE38, and Zn blndkg sits I XNF7) or near the carboxyl terminus (CRAF1, PAS4, CBL, PSC, RAD16, RAG-i, and PAF-l). Several zinc ring domain proteins have ATPase motifs (HIP116, RAD16, and RAD5). One ring protein, MSL-2 (male specific lethal-2) has a metallothionein domain in addition to the zinc ring domain (26). PML belongs to a subfamily of ring proteins that have been shown to possess another cysteine bistidine motif, identified as a â€œB-boxâ€•followingthe zinc ring domain Fig.4. BRCAImodelhighlightingresiduesproposedtoparticipateinBRCA1DNA (30). PML has two B-box domains followed by an a helical, coiled- bindingandprotein-proteininteractions.Residuespredictedtoparticipateinprotein @ coil domain downstream from the zinc ring domain (1 1). The B-box proteininteractions(Lys3t,GInte, Asp67)areshowninred.Residuespredictedtobe ...... involved in DNA binding (GlnM and Lys5Â°) are shown in pink. Zinc ions are shown in motif and additional a helical, coiled-coil domains are absent in @eenCysligands(tozinc)areshownin yellow,andHisligands(tozinc)areshownin BRCA1 and EHV. It has recently been demonstrated that the PML blue.TheCys6'andCysMmissensemutationsitesarelocatedintheZn2 bindingsite. 2541

Table 1 RMS deviationaverageNMR between secondary structural elements of the EHV with the structure. A value of â€”0.4is approximately equivalent to an structureComparison structure and minimized BRC'AI model X-ray crystal structure with a resolution of 2 A (33). NMRstructure of each of the secondary structural elements in the EHV average eachsecondarywith the minimized BRCA1 model structure. By individually comparing WHAT IF checks interatomic distances, atomic packing, torsion antiparallel13structural element of the two molecules, it is possible to see that the and side chain angles, and unsatisfied hydrogen bond donors and duetosheetis wherethetwostructuresexhibitthegreatestdifferenceinstructure.Thisis acceptors and gives a measure of the overall quality of the structure. @ differsfromthe cis-Pro in the @3turnbetween sheets 1and 2 in the EHV structure. This turn @ the more commonly found type I turn in the BRCA1 structure.No. The WHAT IF overall quality score for this BRCA1 model structure was â€”2.8. backboneSecondary of structural RMS deviation atoms superimposed The EHV structure on which this BRCA1 model was based gave a 0)f3sheet element (A) (N, Ca, C, and WHAT IF quality score of â€”2.60.Asis reasonable, the overall quality 40sheet1 1.46 and validity of this model appears comparable to that of the NMR 32Antiparallel2 0.66 structure on which it was based. 72a f3 sheets ((3 sheets 1 and 2) 1.60 72@3sheet3helix 0.69 Molecular Dynamics: Testing the Solvated Structure. Fig. 6 is a 4013 0.71 plot of the backbone RMS deviation versus time across the 140-ps 16813sheets1and2â€”ahelix 1.81 sheets1â€”3-ahelix 1.85 216 trajectory. The backbone RMS deviation of the dynamics structure compared with the starting structure equilibrated between 110 and 140 PS to an average value of 1.48 A. The backbone RMS deviation RMS deviation is 0.018 A (N, Ca, C, and 0). This demonstrates that between the starting and ending structure was 1.65 A. A backbone the zinc ligand distances are maintained without constraints, and that superposition of all seventy structures saved along the molecular the structure does not change significantly when the distance restraints dynamics trajectory shown in Fig. 7 illustrates that there are no major are removed. structural reorganizations along the trajectory. The zinc ligand dis Superimposing the minimized BRCA1 model structure with the tances are maintained within tolerance (Â±0.30 A) along the 140 ps. EHV structure yields a total backbone RMS deviation of 3.0 A. This Overall, the structure is remarkably stable. includes the protein loops, where there is the greatest difference The backbone RMS deviation between the BRCA1 final average between the two structures. A backbone (N, Ca, C, and 0) superpo molecular dynamics structure and the EHV structure is 3.1 A, which sition of the separate secondary structural elements reveals much includes protein loops in the structure. Superposition of the separate greater similarity in the structures (as shown in Table 1). secondary structural elements of these structures, as with the mini Fig. 5 shows a superposition of the minimized BRCA1 model mized BRCA1 structure, also reveals much greater similarity in the structure and EHV average NMR structure. The loop regions of the structures (as shown in Table 2). modeled protein are the most difficult to define, because they exhibit Fig. 5 shows a superposition of the final average molecular dynam the greatest flexibility. The smallest RMS deviations between the ics BRCA1 model structure with the minimized BRCA1 model struc EHV structure and BRCA1 model are in regions of the protein that contain a single, well-defined secondary structure (a helix or single f3 sheet). The greatest difference between the secondary structural dc ments of the minimized BRCAI structure and the EHV structure is in the antiparallel f3 sheet. The @3turn between the two antiparallel (3 sheets differs in both structures. In the EHV structure, this turn (Ala21, Leu22, Pro23, and Cys24) contains a cis-Pro in the i + 2 position and has the characteristic ij and 4 angles of a type V lb (3turn (32). In the BRCA1 model, this turn does not contain a cis-Pro (Thr@7,Lys3t, Cys39, and Asp40) and has the characteristics of the more commonly found type I (3turn. In the loop region ofthe protein [which is between the a helix and the final (3 sheet ((3 sheet 3), consisting of residues 55â€”71in BRCA1I, the BRCA1 model contains two cis-Pro residues (cis-Pro58 and cis-Pro62), the corresponding prolines in the EHV structure are both trans. These prolines must be cis in the BRCA1 structure to accommodate the zinc ligand distances in the structure. The accuracy of the model was validated by subjecting the model structure to PROCHECK version 3.0 (33), and WHAT IF version 4.99 (34). PROCHECK assesses the stereochemical quality of the struc ture, nonbonded contacts within the structure, side-chain conforma tions, bond lengths and angles, and peptide bond planarity (w) and reports the deviations from values in well-refined structures. In the BRCA1 model structure, all 4 and i@,angles fell within allowed regions of the Ramachandran plot. All the main chain parameters (the peptide bond planarity, number of bad nonbonded interactions, hy drogen bond energies, and a-carbon tetrahedral distortion) were all within acceptable limits from ideal values (in well-refined structures). All of the side chain parameters (x' gauche minus and plus and X' trans- and f trans-angle values) were comparable to those for high resolution structures. The overall G-factor value given by PRO CHECK for this structure was â€”0.4.Ideally, scores above (less negative than) â€”0.5 indicate good-quality structures, and values Fig. 5. Superposition of the BRCAI minimized model (yellow), final average molec greater than (more negative than) â€”1.0 are indicative of problems ular dynamics BRCA1 model structure (blue), and EHV average NMR structure (red). 2542

Backbone RMSdeviation vs. time

2 @ 1.8 @ 1.6 1.4 Fig. 6. Plot of the BRCA1 backbone RMS deviation versus 1.2 time for the 140-ps molecular dynamics trajectory. The average structure (110â€”140ps) has a backbone RMS to the starting ! 0.8 structure of 1.48 A. @ 0.6 @ 0.4 @ 0.2 0

Timâ€¢(p.)

nonspecifically to DNA-cellulose (10, 35). Several zinc ring proteins (i.e., ICPO, RO-52K, and PML) have been shown to exhibit nuclear localization (13). It has been suggested that PML is involved in promoting protein-protein interactions that stabilize PML nuclear body formation (11). Other zinc finger motifs in addition to the ring domain that possess two independent zinc-binding sites are the LIM domain and the CRD (36). The CRD domain has interleaved zinc ligands, and its recently solved NMR structure revealed the CRD to have a fold similar to that of the ring domain. Both domains consist of a three-stranded (3 sheet and an a helix. The CRD is in the protein kinase C regulatory domain that is believed to contain the binding sites for diacylglycerol and phorbol esters and to be critical for regulation of kinase activity. The role that the CRD plays, however, is unknown (37). Many of the proteins containing the LIM domain are transcription factors (the name â€œLIMâ€•isderived from the three transcription factors in which it was first observed: Lin-il, Isl-l, and Mec-3. However, like the ring domain, the LIM domain has not been shown to have biologically relevant DNA-binding activity. Recent studies (38) have demonstrated a specific protein-protein interaction between two LIM domain proteins, zyxin and CRP. Therefore, it is possible that the ring Fig. 7. Backbone superposition of all 70 structures along the molecular dynamics trajectory. domain may function similarly to the LIM domain, and that protein protein interactions in which the ring domain participates may play a role in gene expression regulation. Both the LIM and ring domains are tare and EHV average NMR structure. The superposition of the bipartite structures with two zinc-binding domains; therefore, it is average molecular dynamics and minimized BRCA1 model structures possible that they may function similarly to the glucocorticoid recep illustrates that the major region of flexibility in the BRCA1 model tor, in which one zinc site is required for nucleic acid binding, and the structure is the loop region between the a helix and (3sheet 3 (residues other mediates protein-protein interaction, so that the receptor forms 55â€”71).The interleaved zinc ligand structure of the ring domain is a homodimer. This may be the case, because the HSV Vmwl 10 zinc often compared in its ability to stabilize a structure to the cross-linked ring protein has been suggested to be a multimer in solution (35). disulfide bond conformation. This is demonstrated here by the small If BRCA1 functions as a moderator of protein-protein interactions, changes exhibited by the BRCA1 model structure along this molec it may act similarly to the CRD or LIM domain. In the CRD, residues ular dynamics trajectory. (Coordinates for the minimized and average molecular dynamics BRCA1 model structures are available by con tacting the authors.) TableaverageNMR 2 RMS deviation between secondary structural elements of the EHV structureComparisonstructure and the BRCAI final average molecular dynamics NMRstructure of each of the secondary structural elements in the EHV average DISCUSSION eachsecondarywith the BRCA1 final average molecular dynamics structure. By comparing antiparallel13structural element of the two molecules it is possible to see that the A specific common functionality for the zinc ring domain has not exhibitthesheet,asin theminimizedBRCA1modelstructure,iswherethetwostructures been identified to date. The domain appears to interact with DNA greatest difference in structure.No. either through direct binding or indirectly by mediating protein backboneSecondary of structural RMS deviation atoms superimposed protein interactions. No member of the zinc ring domain family has 0)p element (A) (N, C@,C, and been shown to bind DNA in a sequence-dependent manner. Also, the sheet140sheet 1.35 EHV zinc ring domain did not bind to DNA or RNA under assay 32Antiparallel2 0.59 conditions (9). However, a synthetic zinc ring peptide was shown to 72a f3 sheets (@ sheets 1 and 2) 1.38 7213helix 0.60 bind to a subset of randomized DNA sequences, and the DNA-protein 4013sheet3 0.96 complex formation was zinc dependent (6). Likewise, the EHV, 16813sheetsIand2â€”ahelix 1.87 Mel-18, and XNF7 zinc ring domains have been shown to bind sheets1â€”3--ahelix 1.95 216 2543

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1996 American Association for Cancer Research. MOLECULARMODELINGOF BRCAI AMINO-TERMINALZINCRING DOMAIN that are part of the central antiparallel (3 sheet and a helix play a currently linked to breast and/or ovarian cancer. If the BRCA1 zinc significant role in protein binding (37). Although the BRCA1 zinc ring structure is ultimately shown to bind DNA in a manner similar to ring domain possesses only 15% sequence identity and 31% sequence other zinc finger transcription factors, mutations at these positions similarity with CRD, in an alignment by Smith and Waterman (16), may inhibit binding to DNA. Molecular modeling of interactions the central four BRCA1 zinc cysteine ligands, Cys@, Cys47, Cys61, between BRCA1 and DNA will be useful in the design of mutants and Cys@, align with the CRD zinc cysteine ligands Cysâ€•5 Cys'18 aimed at understanding BRCA1 function. Cys'32, and Cys'35. This corresponds to the central antiparallel (3 Several predisposing BRCA1 mutations linked to breast and ovar sheet region of the molecule. Cys'32 and Cys'35 are required for the ian cancer have been identified as occurring in the zinc ring domain. phorbol ester affinity of CRD. Val'@, also required for phorbol ester One of the original BRCA1 mutations (kindred 1901) contains a binding of CRD, is replaced by Leu73 in BRCA1, an aliphatic residue frameshift in exon 2 (l88delll; Ref. 1), which essentially removes the with similar chemical properties. A group of CRD conserved residues zinc ring domain from the protein. In addition, the most common (Tyr'Â°@,Phe'14 Lys131, and Asn'38) have been identified (37) as BRCA1 mutation seen to date is l85delAG (C24ST0P39 in codon 24; being positioned for interactions with protein kinase C activators. In Ref. 45), which essentially disrupts the BRCAJ gene product precisely BRCA1, Tyr'Â°@is replaced by Lys38, another ionizable residue; at the first residue of the C3HC4 domain. Three frequent missense 14 is conserved; Lys13' is replaced by Gln@Â°; and Asn138 is mutations, Cys61Gly, Cys@Gly, and Cys@Tyr; Ref. 45) would also replaced by Asp67. Asn can potentially deamidate to Asp, and these disrupt the zinc ring domain structure. The zinc ion in proteins residues are structurally similar. The Lys38, GlnÂ°Â°,andAsp67 residues interacts strongly with several ligands, the sulfur in Cys, the nitrogen are all on the surface of the BRCA1 zinc ring model and have side in His, and the oxygen in Asp, Glu, and water. If the Cys residues chains with extended orientations favorable for interaction with other were mutated to Gly or Tyr, the zinc ion coordination, no longer proteins (Fig. 4). The Phe43 residue is buried in the BRCA1 model. tetrahedral, would be unstable. The zinc ion would either be lost from These predicted surface residues in BRCA1 (Lys38, Gln@Â°,andAsp67) the structure, and the protein would assume a different fold, or water may participate in protein-protein interactions if the mechanism of would serve as the fourth ligand for zinc, altering the conformation of BRCA1 zinc ring domain protein interaction is similar to that of the the protein. Because the correct protein fold would no longer be CRD. Mutations of these residues would probe the BRCA1 zinc ring maintained, the mutated zinc ring would no longer interact with DNA domain functionality as a mediator of protein-protein interactions. and/or protein in the same manner. Studies are under way to ascertain A variety of zinc finger crystal structures [zif268 (mouse immediate whether the zinc ring domain of BRCA1 can bind DNA in a sequence early protein), human glioblastoma, Gal4 (yeast transcriptional acti specific manner, or whether BRCA1 protein-protein interactions are vator), WT1 , and estrogen and glucocorticoid receptors] have shown of biological significance. interactions between the protein zinc finger a helix and the DNA major groove (39â€”42). Although there are significant differences ACKNOWLEDGMENTS between the zinc ring and zinc finger structures, a comparison be tween the EHV zinc ring and the classic TFIIIA-type ring finger (9) We are grateful to Howard Smith for his assistance and support with showed some structural similarity. Backbone atom superposition of computer graphics. the human enhancer-binding protein TFIIIA-type zinc finger and the EHV zinc ring finger yielded an RMS deviation of 2.0 A (9). 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Rachelle J. Bienstock, Tom Darden, Roger Wiseman, et al.

Cancer Res 1996;56:2539-2545.

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