Skip to content
2000
Volume 32, Issue 9
  • ISSN: 0929-8665
  • E-ISSN: 1875-5305
file format pdf download PDF
file format text download EPUB
side by side viewer icon HTML

Abstract

Introduction

Numerous X-ray crystal structures of the c-Src SH3 domain have provided a large sampling of atomic-level information for this important signaling domain. Multiple crystal forms have been reported, with variable crystal lattice contacts and chemical crystallization conditions.

Materials and Methods

We crystallized the c-Src SH3 domain in a crystallization buffer containing NiCl.

Results

A unique crystal structure of the Src SH3 domain in the trigonal space group 3 is determined to 1.45 Å resolution. Crystal packing and anomalous scattering reveal that this crystal form is mediated by two ordered nickel ions provided by the crystallization buffer. Nickel coordination occurs in a 2:2 stoichiometry, which dimerizes two SH3 domain monomers across a pseudo-twofold rotation axis and involves the native N-terminal c-Src SH3 amino acid sequence, a surface-exposed histidine residue, and ordered water molecules.

Discussion

This study provides an example of metal-mediated crystallization and metal binding by N-terminal protein residues, contrasting with the Amino-Terminal Copper and Nickel Binding (ATCUN) motif.

Conclusion

Alternative avenues help widen the potential for future crystallography-based studies of the c-Src SH3 domain.

© 2025 The Author(s). Published by Bentham Science Publishers.
This is an open access article published under CC BY 4.0 https://creativecommons.org/licenses/by/4.0/legalcode
Loading

Article metrics loading...

/content/journals/ppl/10.2174/0109298665417324250929120040
2025-10-08
2026-02-28

  • From This Site

    /content/journals/ppl/10.2174/0109298665417324250929120040
    dcterms_title,dcterms_subject,pub_keyword
    -contentType:Contributor -contentType:Concept -contentType:Institution
    10
    5
Loading full text...

Full text loading...

/deliver/fulltext/ppl/32/9/PPL-32-9-06.html?itemId=/content/journals/ppl/10.2174/0109298665417324250929120040&mimeType=html&fmt=ahah

References

  1. VogelC. BashtonM. KerrisonN.D. ChothiaC. TeichmannS.A. Structure, function and evolution of multidomain proteins.Curr. Opin. Struct. Biol.200414220821610.1016/j.sbi.2004.03.011 15093836
     [Google Scholar]
  2. KanekoT. LiL. LiS.S. The SH3 domain- a family of versatile peptide- and protein-recognition module.Front. Biosci.2008134938495210.2741/3053 18508559
     [Google Scholar]
  3. MayerB.J. HamaguchiM. HanafusaH. A novel viral oncogene with structural similarity to phospholipase C.Nature1988332616127227510.1038/332272a0 2450282
     [Google Scholar]
  4. MehrabipourM. JasemiN.S.K. DvorskyR. AhmadianM.R. A systematic compilation of human SH3 domains: A versatile superfamily in cellular signaling.Cells20231216205410.3390/cells12162054 37626864
     [Google Scholar]
  5. KazemeinJ.N.S. MehrabipourM. MagdalenaE.E. BrunsveldL. DvorskyR. AhmadianM.R. Functional classification and interaction selectivity landscape of the human SH3 domain superfamily.Cells202413219510.3390/cells13020195 38275820
     [Google Scholar]
  6. NobleM.E. MusacchioA. SarasteM. CourtneidgeS.A. WierengaR.K. Crystal structure of the SH3 domain in human Fyn; Comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin.EMBO J.19931272617262410.1002/j.1460‑2075.1993.tb05922.x 7687536
     [Google Scholar]
  7. YuH. RosenM.K. ShinT.B. Seidel-DuganC. BruggeJ.S. SchreiberS.L. Solution structure of the SH3 domain of Src and identification of its ligand-binding site.Science199225850881665166810.1126/science.1280858 1280858
     [Google Scholar]
  8. MusacchioA. NobleM. PauptitR. WierengaR. SarasteM. Crystal structure of a Src-homology 3 (SH3) domain.Nature1992359639885185510.1038/359851a0 1279434
     [Google Scholar]
  9. TeyraJ. HuangH. JainS. GuanX. DongA. LiuY. TempelW. MinJ. TongY. KimP.M. BaderG.D. SidhuS.S. Comprehensive analysis of the human SH3 domain family reveals a wide variety of non-canonical specificities.Structure201725101598161010.1016/j.str.2017.07.017 28890361
     [Google Scholar]
  10. SakselaK. PermiP. SH3 domain ligand binding: What’s the consensus and where’s the specificity?FEBS Lett.2012586172609261410.1016/j.febslet.2012.04.042 22710157
     [Google Scholar]
  11. ChauJ.E. VishK.J. BoggonT.J. StieglerA.L. SH3 domain regulation of RhoGAP activity: Crosstalk between p120RasGAP and DLC1 RhoGAP.Nat. Commun.2022131478810.1038/s41467‑022‑32541‑4 35970859
     [Google Scholar]
  12. FengS. ChenJ.K. YuH. SimonJ.A. SchreiberS.L. Two binding orientations for peptides to the Src SH3 domain: Development of a general model for SH3-ligand interactions.Science199426651881241124710.1126/science.7526465 7526465
     [Google Scholar]
  13. LimW.A. RichardsF.M. FoxR.O. Structural determinants of peptide-binding orientation and of sequence specificity in SH3 domains.Nature1994372650437537910.1038/372375a0 7802869
     [Google Scholar]
  14. MusacchioA. SarasteM. WilmannsM. High-resolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides.Nat. Struct. Biol.19941854655110.1038/nsb0894‑546 7664083
     [Google Scholar]
  15. YuH. ChenJ.K. FengS. DalgarnoD.C. BrauerA.W. SchrelberS.L. Structural basis for the binding of proline-rich peptides to SH3 domains.Cell199476593394510.1016/0092‑8674(94)90367‑0 7510218
     [Google Scholar]
  16. BoggonT.J. EckM.J. Structure and regulation of Src family kinases.Oncogene200423487918792710.1038/sj.onc.1208081 15489910
     [Google Scholar]
  17. XuW. HarrisonS.C. EckM.J. Three-dimensional structure of the tyrosine kinase c-Src.Nature1997385661759560210.1038/385595a0 9024657
     [Google Scholar]
  18. CloutierJ.F. VeilletteA. Association of inhibitory tyrosine protein kinase p50csk with protein tyrosine phosphatase PEP in T cells and other hemopoietic cells.EMBO J.199615184909491810.1002/j.1460‑2075.1996.tb00871.x 8890164
     [Google Scholar]
  19. AnafiM. RosenM.K. GishG.D. KayL.E. PawsonT. A potential SH3 domain-binding site in the Crk SH2 domain.J. Biol. Chem.199627135213652137410.1074/jbc.271.35.21365 8702917
     [Google Scholar]
  20. SakselaK. ChengG. BaltimoreD. Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4.EMBO J.199514348449110.1002/j.1460‑2075.1995.tb07024.x 7859737
     [Google Scholar]
  21. CicchettiP. MayerB.J. ThielG. BaltimoreD. Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP-rho.Science1992257507180380610.1126/science.1379745 1379745
     [Google Scholar]
  22. KallS.L. WhitlatchK. SmithgallT.E. LavieA. Molecular basis for the interaction between human choline kinase alpha and the SH3 domain of the c-Src tyrosine kinase.Sci. Rep.2019911712110.1038/s41598‑019‑53447‑0 31745227
     [Google Scholar]
  23. LuttrellL.M. FergusonS.S.G. DaakaY. MillerW.E. MaudsleyS. Della RoccaG.J. LinF.T. KawakatsuH. OwadaK. LuttrellD.K. CaronM.G. LefkowitzR.J. Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes.Science1999283540265566110.1126/science.283.5402.655 9924018
     [Google Scholar]
  24. MayerB.J. EckM.J. SH3 Domains: Minding your p’s and q’s.Curr. Biol.19955436436710.1016/S0960‑9822(95)00073‑X 7542990
     [Google Scholar]
  25. BacarizoJ. Martinez-RodriguezS. Martin-GarciaJ.M. Andujar-SanchezM. Ortiz-SalmeronE. NeiraJ.L. Camara-ArtigasA. Electrostatic effects in the folding of the SH3 domain of the c-Src tyrosine kinase: pH-dependence in 3D-domain swapping and amyloid formation.PLoS One2014912e11322410.1371/journal.pone.0113224 25490095
     [Google Scholar]
  26. HegdeR.P. PavithraG.C. DeyD. AlmoS.C. RamakumarS. RamagopalU.A. Can the propensity of protein crystallization be increased by using systematic screening with metals?Protein Sci.20172691704171310.1002/pro.3214 28643473 PMC5563152
     [Google Scholar]
  27. LiutkusM. SasselliI.R. RojasA.L. CortajarenaA.L. Diverse crystalline protein scaffolds through metal‐dependent polymorphism.Protein Sci.2024335e497110.1002/pro.4971 38591647
     [Google Scholar]
  28. Camara-ArtigasA. Plaza-GarridoM. Martinez-RodriguezS. BacarizoJ. New crystal form of human ubiquitin in the presence of magnesium.Acta Crystallogr. F Struct. Biol. Commun.2016721293510.1107/S2053230X15023390 26750481
     [Google Scholar]
  29. LaganowskyA. ZhaoM. SoriagaA.B. SawayaM.R. CascioD. YeatesT.O. An approach to crystallizing proteins by metal‐mediated synthetic symmetrization.Protein Sci.201120111876189010.1002/pro.727 21898649
     [Google Scholar]
  30. QiuX. JansonC.A. Structure of apo acyl carrier protein and a proposal to engineer protein crystallization through metal ions.Acta Crystallogr. D Biol. Crystallogr.20046091545155410.1107/S0907444904015422 15333924
     [Google Scholar]
  31. DuanM. LvC. ZangJ. LengX. ZhaoG. ZhangT. Metals at the helm: Revolutionizing protein assembly and applications.Macromol. Biosci.20242411240012610.1002/mabi.202400126 39239781
     [Google Scholar]
  32. SchneiderD.K. ShiW. AndiB. JakoncicJ. GaoY. BhogadiD.K. MyersS.F. MartinsB. SkinnerJ.M. AishimaJ. QianK. BernsteinH.J. LazoE.O. LangdonT. LaraJ. Shea-McCarthyG. IdirM. HuangL. ChubarO. SweetR.M. BermanL.E. McSweeneyS. FuchsM.R. FMX – The frontier microfocusing macromolecular crystallography beamline at the national synchrotron light source II.J. Synchrotron Radiat.202128265066510.1107/S1600577520016173 33650577
     [Google Scholar]
  33. VonrheinC. FlensburgC. KellerP. SharffA. SmartO. PaciorekW. WomackT. BricogneG. Data processing and analysis with the autoPROC toolbox.Acta Crystallogr. D Biol. Crystallogr.201167429330210.1107/S0907444911007773 21460447
     [Google Scholar]
  34. KabschW. XDS.Acta Crystallogr. D Biol. Crystallogr.201066Pt 212513210.1107/S0907444909047337 20124692 PMC2815665
     [Google Scholar]
  35. EvansP.R. MurshudovG.N. How good are my data and what is the resolution?Acta Crystallogr. D Biol. Crystallogr.20136971204121410.1107/S0907444913000061 23793146
     [Google Scholar]
  36. AfonineP.V. Grosse-KunstleveR.W. EcholsN. HeaddJ.J. MoriartyN.W. MustyakimovM. TerwilligerT.C. UrzhumtsevA. ZwartP.H. AdamsP.D. Towards automated crystallographic structure refinement with phenix.refine.Acta Crystallogr. D Biol. Crystallogr.201268Pt 435236710.1107/S0907444912001308 22505256 PMC3322595
     [Google Scholar]
  37. McCoyA.J. Grosse-KunstleveR.W. AdamsP.D. WinnM.D. StoroniL.C. ReadR.J. Phaser crystallographic software.J. Appl. Cryst.200740465867410.1107/S0021889807021206 19461840
     [Google Scholar]
  38. TerwilligerT.C. Grosse-KunstleveR.W. AfonineP.V. MoriartyN.W. ZwartP.H. HungL.W. ReadR.J. AdamsP.D. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard.Acta Crystallogr. D Biol. Crystallogr.200864Pt 1616910.1107/S090744490705024X 18094468 PMC2394820
     [Google Scholar]
  39. EmsleyP. CowtanK. Coot: Model-building tools for molecular graphics.Acta. Crystallogr D Biol. Crystallogr200460Pt 12 Pt 12126213210.1107/S0907444904019158 15572765
     [Google Scholar]
  40. KabschW. SanderC. Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features.Biopolymers198322122577263710.1002/bip.360221211 6667333
     [Google Scholar]
  41. GucwaM. LenkiewiczJ. ZhengH. CymborowskiM. CooperD.R. MurzynK. MinorW. CMM —An enhanced platform for interactive validation of metal binding sites.Protein Sci.2023321e452510.1002/pro.4525 36464767
     [Google Scholar]
  42. LiuQ. LiuQ. HendricksonW.A. Robust structural analysis of native biological macromolecules from multi-crystal anomalous diffraction data.Acta Crystallogr. D Biol. Crystallogr.20136971314133210.1107/S0907444913001479 23793158
     [Google Scholar]
  43. El OmariK. ForsythI. DumanR. OrrC.M. MykhaylykV. ManciniE.J. WagnerA. Utilizing anomalous signals for nlm identification in macromolecular crystallography.Acta Crystallogr. D Struct. Biol.2024801071372110.1107/S2059798324008659 39291627
     [Google Scholar]
  44. SasakiS. Numerical tables of anomalous scattering factors calculated by the cromer and Liberman’s method.1989Available from: https://inis.iaea.org/records/yv4rm-p6961
    [Google Scholar]
  45. McNicholasS. PottertonE. WilsonK.S. NobleM.E.M. Presenting your structures: The CCP 4 mg molecular-graphics software.Acta Crystallogr. D Biol. Crystallogr.201167438639410.1107/S0907444911007281 21460457
     [Google Scholar]
  46. PettersenE.F. GoddardT.D. HuangC.C. MengE.C. CouchG.S. CrollT.I. MorrisJ.H. FerrinT.E. UCSF ChimeraX: Structure visualization for researchers, educators, and developers.Protein Sci.2021301708210.1002/pro.3943 32881101
     [Google Scholar]
  47. MorinA. EisenbraunB. KeyJ. SanschagrinP.C. TimonyM.A. OttavianoM. SlizP. Collaboration gets the most out of software.eLife20132e0145610.7554/eLife.01456 24040512
     [Google Scholar]
  48. KrissinelE. HenrickK. Inference of macromolecular assemblies from crystalline state.J. Mol. Biol.2007372377479710.1016/j.jmb.2007.05.022 17681537
     [Google Scholar]
  49. KrissinelE. HenrickK. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions.Acta. Crystallogr D Biol. Crystallogr200460Pt 12 Pt 12256226810.1107/S0907444904026460 15572779
     [Google Scholar]
  50. HolmL. RosenströmP. Dali server: Conservation mapping in 3D.Nucleic Acids Res.201038Web Server issueW545W54910.1093/nar/gkq366 20457744 PMC2896194
     [Google Scholar]
  51. AndreiniC. CavallaroG. LorenziniS. FindGeo: A tool for determining metal coordination geometry.Bioinformatics201228121658166010.1093/bioinformatics/bts246 22556364
     [Google Scholar]
  52. OpdamL.V. PolancoE.A. de RegtB. LambertinaN. BakkerC. BonnetS. PanditA. A screening method for binding synthetic metallo-complexes to haem proteins.Anal. Biochem.202265311478810.1016/j.ab.2022.114788 35732212
     [Google Scholar]
  53. NowakowskiA.B. WobigW.J. PeteringD.H. Native SDS-PAGE: High resolution electrophoretic separation of proteins with retention of native properties including bound metal ions.Metallomics2014651068107810.1039/C4MT00033A 24686569
     [Google Scholar]
  54. SankararamakrishnanR. VermaS. KumarS. ATCUN‐like metal‐binding motifs in proteins: Identification and characterization by crystal structure and sequence analysis.Proteins200558121122110.1002/prot.20265 15508143
     [Google Scholar]
  55. LaussacJ.P. SarkerB. Characterization of the copper(II) and nickel(II) transport site of human serum albumin. Studies of copper(II) and nickel(II) binding to peptide 1-24 of human serum albumin by carbon-13 and proton NMR spectroscopy.Biochemistry198423122832283810.1021/bi00307a046 6547847
     [Google Scholar]
  56. MaitiB.K. GovilN. KunduT. MouraJ.J.G. Designed metal-ATCUN derivatives: Redox- and non-redox-based applications relevant for chemistry, biology, and medicine.iScience2020231210179210.1016/j.isci.2020.101792 33294799
     [Google Scholar]
  57. KotuniakR. BalW. Kinetics of Cu(ii) complexation by ATCUN/NTS and related peptides: A gold mine of novel ideas for copper biology.Dalton Trans.2021511142610.1039/D1DT02878B 34816848
     [Google Scholar]
  58. Plaza-GarridoM. Salinas-GarcíaM.C. MartínezJ.C. Cámara-ArtigasA. The effect of an engineered ATCUN motif on the structure and biophysical properties of the SH3 domain of c-Src tyrosine kinase.J. Biol. Inorg. Chem.202025462163410.1007/s00775‑020‑01785‑0 32279137
     [Google Scholar]
  59. BacarizoJ. Camara-ArtigasA. Atomic resolution structures of the c-Src SH3 domain in complex with two high-affinity peptides from classes I and II.Acta Crystallogr. D Biol. Crystallogr.201369575676610.1107/S0907444913001522 23633584
     [Google Scholar]
  60. DaviesT.G. TickleI.J. Fragment screening using X-ray crystallography.Top. Curr. Chem.2011317335910.1007/128_2011_179 21678136
     [Google Scholar]
  61. PatelD. BaumanJ.D. ArnoldE. Advantages of crystallographic fragment screening: Functional and mechanistic insights from a powerful platform for efficient drug discovery.Prog. Biophys. Mol. Biol.20141162-39210010.1016/j.pbiomolbio.2014.08.004 25117499
     [Google Scholar]
  62. GoldschmidtL. CooperD.R. DerewendaZ.S. EisenbergD. Toward rational protein crystallization: A web server for the design of crystallizable protein variants.Protein Sci.20071681569157610.1110/ps.072914007 17656576
     [Google Scholar]
  63. WalterT.S. MeierC. AssenbergR. AuK.F. RenJ. VermaA. NettleshipJ.E. OwensR.J. StuartD.I. GrimesJ.M. Lysine methylation as a routine rescue strategy for protein crystallization.Structure200614111617162210.1016/j.str.2006.09.005 17098187
     [Google Scholar]
  64. ForseG.J. RamN. BanataoD.R. CascioD. SawayaM.R. KlockH.E. LesleyS.A. YeatesT.O. Synthetic symmetrization in the crystallization and structure determination of CelA from Thermotoga maritima.Protein Sci.201120116817810.1002/pro.550 21082721 PMC3047073
     [Google Scholar]
  65. BanataoD.R. CascioD. CrowleyC.S. FleissnerM.R. TiensonH.L. YeatesT.O. An approach to crystallizing proteins by synthetic symmetrization.Proc. Natl. Acad. Sci. USA200610344162301623510.1073/pnas.0607674103 17050682
     [Google Scholar]
  66. YamadaH. TamadaT. KosakaM. MiyataK. FujikiS. TanoM. MoriyaM. YamanishiM. HonjoE. TadaH. InoT. YamaguchiH. FutamiJ. SenoM. NomotoT. HirataT. YoshimuraM. KurokiR. ‘Crystal lattice engineering,’ an approach to engineer protein crystal contacts by creating intermolecular symmetry: Crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines.Protein Sci.20071671389139710.1110/ps.072851407 17586772
     [Google Scholar]
  67. SalgadoE.N. RadfordR.J. TezcanF.A. Metal-directed protein self-assembly.Acc. Chem. Res.201043566167210.1021/ar900273t 20192262
     [Google Scholar]
  68. TrakhanovS. QuiochoF.A. Influence of divalent cations in protein crystallization.Protein Sci.1995491914191910.1002/pro.5560040925 8528088
     [Google Scholar]
  69. HandingK.B. NiedzialkowskaE. ShabalinI.G. KuhnM.L. ZhengH. MinorW. Characterizing metal-binding sites in proteins with X-ray crystallography.Nat. Protoc.20181351062109010.1038/nprot.2018.018 29674755
     [Google Scholar]
  70. PakharukovaN. ThomasB.N. BansiaH. LiL. AbzalimovR.R. KimJ. KahsaiA.W. PaniB. BassfordD.K. LiuS. ZhangX. des GeorgesA. LefkowitzR.J. Beta-arrestin 1 mediated Src activation via Src SH3 domain revealed by cryo-electron microscopy.bioRxiv20243913140210.1101/2024.07.31.605623
     [Google Scholar]
  71. Longshore-NeateF. CeravoloC. MasugaC. TahtiE.F. BlountJ.M. SmithS.N. AmacherJ.F. The conformation of the nSrc specificity-determining loop in the Src SH3 domain is modulated by a WX conserved sequence motif found in SH3 domains.Front. Mol. Biosci.202411148727610.3389/fmolb.2024.1487276 39698111
     [Google Scholar]
  72. ChenV.B. ArendallW.B. HeaddJ.J. KeedyD.A. ImmorminoR.M. KapralG.J. MurrayL.W. RichardsonJ.S. RichardsonD.C. MolProbity: All-atom structure validation for macromolecular crystallography.Acta Crystallogr. D Biol. Crystallogr.201066Pt 1122110.1107/S0907444909042073 20057044 PMC2803126
     [Google Scholar]
/content/journals/ppl/10.2174/0109298665417324250929120040
Loading
Nickel Binding to the c-Src SH3 Domain Facilitates Crystallization
PPL 32, 679 (2025); https://doi.org/10.2174/0109298665417324250929120040
/content/journals/ppl/10.2174/0109298665417324250929120040
/content/journals/ppl/10.2174/0109298665417324250929120040
Loading

Data & Media loading...

Supplements

1. Supplementary Tables, Figures, and Figure Legends file (Calicdan_Stiegler_PPL_submission-supplemen-tary.docx). 2. PDB submission validation report file (Calicdan-Stiegler-PDB-9OFX-Validation-Report.pdf). 3. Expression plasmid map file (Calicdan-Stiegler-hSrc-SH3NH_85-143_pET28a_Map.tiff). 4. Expression plasmid full sequence file (Calicdan-Stiegler-hSrc-SH3NH_85-143_pET28a.txt). Supplementary material is available on the publisher’s website along with the published article.

file format download Download

  • Article Type:     Research Article
Keyword(s): anomalous signal; c-Src tyrosine kinase; crystal structure; dimerization; nickel coordination; SH3 domain

Most Cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error
Please enter a valid_number test