Comprehensive macromolecular conformations mapped by quantitative SAXS analyses

Abstract

Comprehensive perspectives of macromolecular conformations are required to connect structure to biology. Here we present a small angle X-ray scattering (SAXS) Structural Similarity Map (SSM) and Volatility of Ratio (V_R) metric providing comprehensive, quantitative and objective (superposition-independent) perspectives on solution state conformations. We validate V_R and SSM utility on human MutSβ, a key ABC ATPase and chemotherapeutic target, by revealing MutSβ DNA sculpting and identifying multiple conformational states for biological activity.

Figure 1

SAXS as a measure of structural similarity; scored by three metrics (Chi², Pearson, and V_R). Scores were assigned a gradient color with white - low agreement and red - high agreement. Right-most box in each panel are self-comparisons. (a) Phosphorylation state modulates the coupling of domains in Retinoblastoma protein. Snapshots from a molecular dynamics simulation of this process were taken (right to left) 1000, 2000, 4000, 8000 and 16000 time steps away from the reference structure. The closer in time the snapshots are, the more correlated their structure. (b) Aspartate Transcarbamoylase (ATCase) is a canonical cooperative enzyme with available high resolution structures of a tense state (T), relaxed state (R) and mutant intermediate. Calculated SAXS curves from the three structures and mixtures of T and R states were judged for agreement against reference structure T-state (c) Comparing a protein structure against (right to left) another with identical number of amino acids but different fold, their dimers, and 25% truncations. *indicates where the trends in metrics disagree.

Figure 2

A SAXS structural similarity map (SSM) for MutSβ with and without ADP, ATP, ATP analogs and DNA. A schematic of the complex is shown in a). The SAXS SSM is shown in b). The diagonal (black squares) compares a SAXS curve to itself. * compares states with vs without DNA c) Non-denaturing gel electrophoresis of the samples show DNA binding. (d) Ultraviolet crosslinking following ATP hydrolysis.