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bioRxiv · 10.1101/2025.05.08.652639

Nickel binding to a split ATCUN motif in c-Src SH3 domain facilitates crystallization

Calicdan, X.·Fisher, O. S.·Ha, B. H.·Boggon, T. J.·Stiegler, A. L.
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Abstract

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. Here, we report a unique crystal structure of Src SH3 domain in trigonal space group H32 to 1.45 [A] 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. This study provides an example of metal binding by N-terminal protein residues that contrasts the amino terminal copper and nickel binding (ATCUN) motif and is an alternative avenue for crystallization of the Src SH3 domain. STRUCTURED ABSTRACTO_ST_ABSIntroductionC_ST_ABSNumerous 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 MethodsWe crystallize the c-Src SH3 domain in a crystallization buffer containing NiCl2. ResultsA unique crystal structure of Src SH3 domain in trigonal space group H32 to 1.45 [A] resolution is determined. 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. DiscussionThis study provides an example of metal-mediated crystallization and metal binding by N-terminal protein residues that contrasts the amino terminal copper and nickel binding (ATCUN) motif. ConclusionAlternative avenues for helps widen the potential for future crystallography-based studies of the c-Src SH3 domain.

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Calicdan, X., Fisher, O. S., Ha, B. H., Boggon, T. J., Stiegler, A. L.. 2025-05-13. Nickel binding to a split ATCUN motif in c-Src SH3 domain facilitates crystallization. https://doi.org/10.1101/2025.05.08.652639

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