Development of a structure-analysis pipeline using multiple-solvent crystal structures of barrier-to-autointegration factor.

The multiple-solvent crystal structure (MSCS) approach uses high concentrations of organic solvents to characterize the interactions and effects of solvents on proteins. Here, the method has been further developed and an MSCS data-handling pipeline is presented that uses the Detection of Related Solvent Positions (DRoP) program to improve data quality. DRoP is used to selectively model conserved water molecules, so that an advanced stage of structural refinement is reached quickly. This allows the placement of organic molecules more accurately and convergence on high-quality maps and structures. This pipeline was applied to the chromatin-associated protein barrier-to-autointegration factor (BAF), resulting in structural models with better than average statistics. DRoP and Phenix Structure Comparison were used to characterize the data sets and to identify a binding site that overlaps with the interaction site of BAF with emerin. The conserved water-mediated networks identified by DRoP suggested a mechanism by which water molecules are used to drive the binding of DNA. Normalized and differential B-factor analysis is shown to be a valuable tool to characterize the effects of specific solvents on defined regions of BAF. Specific solvents are identified that cause stabilization of functionally important regions of the protein. This work presents tools and a standardized approach for the analysis and comprehension of MSCS data sets.

Multiple solvent crystal structures of ribonuclease A: an assessment of the method.

The multiple solvent crystal structures (MSCS) method uses organic solvents to map the surfaces of proteins. It identifies binding sites and allows for a more thorough examination of protein plasticity and hydration than could be achieved by a single structure. The crystal structures of bovine pancreatic ribonuclease A (RNAse A) soaked in the following organic solvents are presented: 50% dioxane, 50% dimethylformamide, 70% dimethylsulfoxide, 70% 1,6-hexanediol, 70% isopropanol, 50% R,S,R-bisfuran alcohol, 70% t-butanol, 50% trifluoroethanol, or 1.0M trimethylamine-N-oxide. This set of structures is compared with four sets of crystal structures of RNAse A from the protein data bank (PDB) and with the solution NMR structure to assess the validity of previously untested assumptions associated with MSCS analysis. Plasticity from MSCS is the same as from PDB structures obtained in the same crystal form and deviates only at crystal contacts when compared to structures from a diverse set of crystal environments. Furthermore, there is a good correlation between plasticity as observed by MSCS and the dynamic regions seen by NMR. Conserved water binding sites are identified by MSCS to be those that are conserved in the sets of structures taken from the PDB. Comparison of the MSCS structures with inhibitor-bound crystal structures of RNAse A reveals that the organic solvent molecules identify key interactions made by inhibitor molecules, highlighting ligand binding hot-spots in the active site. The present work firmly establishes the relevance of information obtained by MSCS.