Structure and Dynamics of the S100A5-RAGE complex and anti-CRISPR AcrIIA4 probed by NMR spectroscopy

Abstract

Distinct protein-protein interactions were investigated using NMR spectroscopy as well biophysical characterization tools such as calorimetry and size exclusion chromatography. In Chapter I, calcium binding of S100A5 and the interaction between S100A5 and receptor for advanced glycation end product (RAGE) were investigated using calorimetry and NMR spectroscopy. S100A5 exhibits a sequential manner to bind calcium ions using the C-terminal and N-terminal calcium binding EF-hands with the equilibrium dissociation constants (KD) of 1.3 µM and 3.5 µM, respectively. S100A5 interacts with the V domain of RAGE (RAGEv) to form a heterotrimeric complex with KD of 5.9 µM in a calcium-dependent manner, which is a distinct stoichiometry from the S100 family proteins. Chemical shift perturbation data from NMR titration experiments indicates that S100A5 employs the periphery of the dimer interface to interact with RAGEv. Distinct binding mode and stoichiometry of against different S100 family proteins could be important to modulate diverse RAGE signaling. In Chapter II, solution structure and dynamics of AcrIIA4 that inhibits Streptococcus pyogenes Cas9 (SpyCas9) widely used for genome editing were investigated. The structure of AcrIIA4 is a compact monomeric αβββαα fold consisting of three α helices flanked against three antiparallel β strands and short 310 helix. The backbone dynamics of AcrIIA4 reveals that distinct backbone dynamics in fast and slow timescales at loop regions is related to interaction surface for SpyCas9. Furthermore, AcrIIA4 binds to apo-SpyCas9 with KD ~4.8 µM and ~0.6 nM for AcrIIA4 binding to sgRNA-bound SpyCas9, which infers that a binary complex between AcrIIA4 and SpyCas9 can associate with sgRNA to form a tight ternary complex, avoiding competition with the DNA substrate for SpyCas9 binding. This studies provide insights into anti-CRISPR-mediated inhibition mechanisms for disabling the SpyCas9, thereby broadening CRISPR-Cas regulatory tools for genome editing.