Perspectives on solution-based small angle X-ray scattering for protein and biological macromolecule structural biology.

Small-angle X-ray scattering (SAXS) is used to extract structural information from a wide variety of non-crystalline samples in different fields (e.g., materials science, physics, chemistry, and biology). This review provides an overview of SAXS as applied to structural biology, specifically for proteins and other biomacromolecules in solution with an emphasis on extracting key structural parameters and the interpretation of SAXS data using a diverse array of techniques. These techniques cover aspects of building and assessing models to describe data measured from monodispersed and ideal dilute samples through to more complicated structurally polydisperse systems. Ab initio modelling, rigid body modelling as well as normal-mode analysis, molecular dynamics, mixed component and structural ensemble modelling are discussed. Dealing with polydispersity both physically in terms of component separation as well as approaching the analysis and modelling of data of mixtures and evolving systems are described, including methods for data decomposition such as single value decomposition/principle component analysis and evolving factor analysis. This review aims to highlight that solution SAXS, with the cohort of developments in data analysis and modelling, is well positioned to build upon the traditional 'single particle view' foundation of structural biology to take the field into new areas for interpreting the structures of proteins and biomacromolecules as population-states and dynamic structural systems.

Determinants of receptor tyrosine phosphatase homophilic adhesion: Structural comparison of PTPRK and PTPRM extracellular domains.

Type IIB receptor protein tyrosine phosphatases are cell surface transmembrane proteins that engage in cell adhesion via their extracellular domains (ECDs) and cell signaling via their cytoplasmic phosphatase domains. The ECDs of type IIB receptor protein tyrosine phosphatases form stable, homophilic, and trans interactions between adjacent cell membranes. Previous work has demonstrated how one family member, PTPRM, forms head-to-tail homodimers. However, as the interface was composed of residues conserved across the family, the determinants of homophilic specificity remain unknown. Here, we have solved the X-ray crystal structure of the membrane-distal N-terminal domains of PTPRK that form a head-to-tail dimer consistent with intermembrane adhesion. Comparison with the PTPRM structure demonstrates interdomain conformational differences that may define homophilic specificity. Using small-angle X-ray scattering, we determined the solution structures of the full-length ECDs of PTPRM and PTPRK, identifying that both are rigid extended molecules that differ in their overall long-range conformation. Furthermore, we identified one residue, W351, within the interaction interface that differs between PTPRM and PTPRK and showed that mutation to glycine, the equivalent residue in PTPRM, abolishes PTPRK dimer formation in vitro. This comparison of two members of the receptor tyrosine phosphatase family suggests that homophilic specificity is driven by a combination of shape complementarity and specific but limited sequence differences.

Structural, Thermodynamic and Enzymatic Characterization of N,N-Diacetylchitobiose Deacetylase from Pyrococcus chitonophagus.

Chitin is a major source of energy and macroelements for many organisms. An important step in its degradation is the deacetylation of chitin or its fragments. Deacetylase from the extremophile Pyrococcus chitonophagus has been analyzed by X-ray crystallography, small-angle X-ray scattering, differential scanning calorimetry, isothermal titration calorimetry and NMR to determine its structure, thermodynamics and enzymatic properties. It is a hexameric, zinc-containing metalloenzyme that retains its structural integrity up to temperatures slightly exceeding 100 °C. It removes the acetyl group specifically from the non-reducing end of the sugar substrate. Its main substrate is N,N-diacetylchitobiose but it also active, at a reduced level, toward N-acetyl-d-glucosamine or a trimer of N-acetyl-d-glucosamine units. Crystallographic analysis includes the structure of the enzyme with its main substrate approaching the active site in a monodentate manner, replacing the single water molecule that is bound at the Zn2+ cation when the ligand is absent. The Zn2+ cation remains tetrahedrally coordinated, with three of its ligands provided by the protein's conserved His-Asp-His triad. The crystal structures are consistent with the reaction mechanism proceeding via an anhydride intermediate. Hydrolysis as the first step cannot be ruled out in a hydrated environment but no defined 'hydrolytic water' site can be identified in the analyzed structures.