Solvent organization is a key but underexploited contributor to the thermodynamics of protein−ligand recognition, with implications for ligand discovery, drug resistance, and protein engineering. Here, we explore the contribution of solvent to ligand binding in the Haemophilus influenzae virulence protein SiaP. By introducing a single mutation without direct ligand contacts, we observed a >1000-fold change in sialic acid binding affinity. Crystallographic and calorimetric data of wild-type and mutant SiaP showed that this change results from an enthalpically unfavorable perturbation of the solvent network. This disruption is reflected by changes in the normalized atomic displacement parameters of crystallographic water molecules. In SiaP's enclosed cavity, relative differences in water-network dynamics serve as a simple predictor of changes in the free energy of binding upon changing protein, ligand, or both. This suggests that solvent structure is an evolutionary constraint on protein sequence that contributes to ligand affinity and selectivity.

Background The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. Methodology and Principal Findings SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes. Significance This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex.

Fragment-based approaches are used routinely to discover enzyme inhibitors as cellular tools and potential therapeutic agents. There have been few reports, however, of the discovery of small-molecule enzyme activators. Herein, we describe the discovery and characterization of small-molecule activators of a glycoside hydrolase (a bacterial O-GlcNAc hydrolase). A ligand-observed NMR screen of a library of commercially available fragments identified an enzyme activator which yielded an approximate 90 % increase in kcat/KM values (kcat=catalytic rate constant; KM=Michaelis constant). This compound binds to the enzyme in close proximity to the catalytic center. Evolution of the initial hits led to improved compounds that behave as nonessential activators effecting both KM and Vmax values (Vmax=maximum rate of reaction). The compounds appear to stabilize an active “closed” form of the enzyme. Such activators could offer an orthogonal alternative to enzyme inhibitors for perturbation of enzyme activity in vivo, and could also be used for glycoside hydrolase activation in many industrial processes.

Structure-guided tethering of a fragment activator significantly increases enzyme activity.

α-Mannoside β-1,6-N-acetylglucos­aminyl­transferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed. Despite intense interest, the catalytic mechanism of MGAT5 is not known in detail, precluding therapeutic exploitation. We solved structures of MGAT5 complexed to glycosyl donor and acceptor ligands, revealing an unforeseen role for donor-induced loop rearrangements in controlling acceptor substrate engagement. QM/MM metadynamics simulations of MGAT5 catalysis highlight the key assisting role of Glu297 and reveal considerable conformational distortions imposed upon the glycosyl donor during transfer. Detailed mechanistic characterization of MGAT5 will aid inhibitor development to correct cancer-associated N-glycosylation.