Hsp100 chaperones cooperate with the Hsp70 chaperone system to disaggregate and reactivate heat-denatured aggregated proteins to promote cell survival after heat stress. The homology models of Hsp100 disaggregases suggest the presence of a conserved network of ionic interactions between the first nucleotide binding domain (NBD1) and the coiled-coil middle subdomain, the signature domain of disaggregating chaperones. Mutations intended to disrupt the putative ionic interactions in yeast Hsp104 and bacterial ClpB disaggregases resulted in remarkable changes of their biochemical properties. These included an increase in ATPase activity, a significant increase in the rate of in vitro substrate renaturation, and partial independence from the Hsp70 chaperone in disaggregation. Paradoxically, the increased activities resulted in serious growth impediments in yeast and bacterial cells instead of improvement of their thermotolerance. Our results suggest that this toxic activity is due to the ability of the mutated disaggregases to unfold independently from Hsp70, native folded proteins. Complementary changes that restore particular salt bridges within the suggested network suppressed the toxic effects. We propose a novel structural aspect of Hsp100 chaperones crucial for specificity and efficiency of the disaggregation reaction.

Summary: Participating as the Cornell-Gdansk group, we have used our physics-based coarse-grained UNited RESidue (UNRES) force field to predict protein structure in the 11th Community Wide Exercise on the Critical Assessment of Techniques for Protein Structure Prediction (CASP11). Our methodology involved extensive multiplexed replica exchange simulations of the target proteins with a recently-improved UNRES force field to provide better reproductions of the local structures of polypeptide chains. All simulations were started from fully-extended polypeptide chains, and no external information was included in the simulation process except for weak restraints on secondary structure to enable us to finish each prediction within the allowed three-week time window. Because of simplified UNRES representation of polypeptide chains, use of enhanced sampling methods, code optimization and parallelization and sufficient computational resources, we were able to treat, for the first time, all 55 human prediction targets with sizes from 44 to 595 amino-acid residues, the average size being 251 residues. Complete structures of six single-domain proteins were predicted accurately, with the highest accuracy being attained for the T0769, for which the CαRMSD was 3.8 Å for 97 residues of the experimental structure. Correct structures were also predicted for 13 domains of multi-domain proteins with accuracy comparable to that of the best template based modeling (TBM) methods. With further improvements of the UNRES force field that are now underway, our physics-based coarse-grained approach to protein-structure prediction will eventually reach global prediction capacity and, consequently, reliability in simulating protein structure and dynamics that are important in biochemical processes.

Oxytocin (OT), synthesized in the heart, has the ability to heal injured hearts and to promote cardiomyogenesis from stem cells. Recently, we reported that the OT-GKR molecule, a processing intermediate of OT, potently increased the spontaneous formation of cardiomyocytes (CM) in embryonic stem D3 cells and augmented glucose uptake in newborn rat CM above the level stimulated by OT. In the present experiments, we investigated whether OT-GKR exists in fetal and newborn rodent hearts, interacts with the OT receptors (OTR) and primes the generation of contracting cells expressing CM markers in P19 cells, a model for the study of early heart differentiation. High performance liquid chromatography of newborn rat heart extracts indicated that OT-GKR was a dominant form of OT. Immunocytochemistry of mouse embryos (embryonic day 15) showed cardiac OT-GKR accumulation and OTR expression. Computerized molecular modeling revealed OT-GKR docking to active OTR sites and to V1a receptor of vasopressin. In embryonic P19 cells, OT-GKR induced contracting cell colonies and ventricular CM markers more potently than OT, an effect being suppressed by OT antagonists and OTR-specific small interfering (si) RNA. The V1a receptor antagonist and specific si-RNA also significantly reduced OT-GKR-stimulated P19 contracting cells. In comparison to OT, OT-GKR induced in P19 cells less α-actinin, myogenin and MyoD mRNA, skeletal muscle markers. These results raise the possibility that C-terminally extended OT molecules stimulate CM differentiation and contribute to heart growth during fetal life.