3NSK

Crystal Structure of the Post-Refolded S100A3 R51A Mutant Expressed in Insect Cell

Summary for 3NSK

• Entry DOI: 10.2210/pdb3nsk/pdb
• Related: 3NSI 3NSL 3NSO
• Descriptor: Protein S100-A3 (2 entities in total)
• Functional Keywords: ef-hand, ca2+, zn2+-binding, metal binding protein
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 2
• Total formula weight: 23278.60

Authors

Unno, M.,Takahara, H.,Kizawa, K. (deposition date: 2010-07-01, release date: 2011-03-16, Last modification date: 2024-10-30)

Primary citation

Unno, M.,Kawasaki, T.,Takahara, H.,Heizmann, C.W.,Kizawa, K.
Refined crystal structures of human Ca(2+)/Zn(2+)-binding S100A3 protein characterized by two disulfide bridges
J.Mol.Biol., 408:477-490, 2011

PubMed Abstract: S100A3, a member of the EF-hand-type Ca(2+)-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn(2+) affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca(2+)/Zn(2+) supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A+C68A) abolished the Ca(2+) binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca(2+) affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A+C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15-1.40 Å resolution shows that SS1 renders the C-terminal classical Ca(2+)-binding loop flexible, which are essential for its Ca(2+) binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn(2+) by (Cys)(3)His residues that is compatible with SS2 formation in S100A3.

PubMed: 21377473
DOI: 10.1016/j.jmb.2011.02.055

Experimental method

X-RAY DIFFRACTION (1.55 Å)