8T8G

C208A Streptococcus pyogenes Sortase A (spySrtA) bound to LPALA peptide

8T8G の概要

• エントリーDOI: 10.2210/pdb8t8g/pdb
• 分子名称: Sortase, LEU-PRO-ALA-LEU-ALA-GLY (3 entities in total)
• 機能のキーワード: sortase-fold, hydrolase, sortase, eight-stranded beta barrel, transpeptidase, housekeeping sortase, surface protein
• 由来する生物種: Streptococcus pyogenes; 詳細
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 19129.70

構造登録者

Kodama, H.M.,Amacher, J.F. (登録日: 2023-06-22, 公開日: 2024-03-13)

主引用文献

Vogel, B.A.,Blount, J.M.,Kodama, H.M.,Goodwin-Rice, N.J.,Andaluz, D.J.,Jackson, S.N.,Antos, J.M.,Amacher, J.F.
A unique binding mode of P1' Leu-containing target sequences for Streptococcus pyogenes sortase A results in alternative cleavage.
Rsc Chem Biol, 5:30-40, 2024

PubMed Abstract: Sortase enzymes are cysteine transpeptidases that attach environmental sensors, toxins, and other proteins to the cell surface in Gram-positive bacteria. The recognition motif for many sortases is the cell wall sorting signal (CWSS), LPXTG, where X = any amino acid. Recent work from ourselves and others has described recognition of additional amino acids at a number of positions in the CWSS, specifically at the Thr (or P1) and Gly (or P1') positions. In addition, although standard cleavage occurs between these two residues (P1/P1'), we previously observed that the SrtA enzyme from will cleave after the P1' position when its identity is a Leu or Phe. The stereochemical basis of this alternative cleavage is not known, although homologs, , SrtA from or do not show alternative cleavage to a significant extent. Here, we use protein biochemistry, structural biology, and computational biochemistry to predict an alternative binding mode that facilitates alternative cleavage. We use SrtA (spySrtA) as our model enzyme, first confirming that it shows similar standard/alternative cleavage ratios for LPAT, LPAT, and LPAT sequences. Molecular dynamics simulations suggest that when P1' is Leu, this amino acid binds in the canonical S1 pocket, pushing the P1 Thr towards solvent. The P4 Leu (L̲PATL) binds as it does in standard binding, resulting in a puckered binding conformation. We use P1 Glu-containing peptides to support our hypotheses, and present the complex structure of spySrtA-LPALA to confirm favorable accommodation of Leu in the S1 pocket. Overall, we structurally characterize an alternative binding mode for spySrtA and specific target sequences, expanding the potential protein engineering possibilities in sortase-mediated ligation applications.

PubMed: 38179192
DOI: 10.1039/d3cb00129f

実験手法

X-RAY DIFFRACTION (1.5 Å)