6 research outputs found
Switch I closure simultaneously promotes strong binding to actin and ADP in smooth muscle myosin
The motor protein myosin uses energy derived from ATP hydrolysis to produce force and motion. Important conserved components (P-loop, switch I, and switch II) help propagate small conformational changes at the active site into large scale conformational changes in distal regions of the protein. Structural and biochemical studies have indicated that switch I may be directly responsible for the reciprocal opening and closing of the actin and nucleotide-binding pockets during the ATPase cycle, thereby aiding in the coordination of these important substrate-binding sites. Smooth muscle myosin has displayed the ability to simultaneously bind tightly to both actin and ADP, although it is unclear how both substrate-binding clefts could be closed if they are rigidly coupled to switch I. Here we use single tryptophan mutants of smooth muscle myosin to determine how conformational changes in switch I are correlated with structural changes in the nucleotide and actin-binding clefts in the presence of actin and ADP. Our results suggest that a closed switch I conformation in the strongly bound actomyosin-ADP complex is responsible for maintaining tight nucleotide binding despite an open nucleotide-binding pocket. This unique state is likely to be crucial for prolonged tension maintenance in smooth muscle
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Design of amyloidogenic peptide traps.
Acknowledgements: We thank the Institute for Protein Design and Baker laboratory members for general discussion and in particular Y. Kipnis, I. Goreshnik, W. Yang, G.-R. Lee and S. Pellock for helpful advice, P. Salveson and L. Stewart for discussions on amyloidogenic peptides and neurodegenerative diseases, N. Ennist for assistance with circular dichroism spectroscopy, B.I.M. Wicky and L. Milles at the Institute for Protein Design for providing Golden Gate cloning vectors and L. Carter for assistance with international shipping. We also thank A. Gonzalez-Diaz under supervision of M. Vendruscolo for her help and expert advice in the cell toxicity assays. This work was supported by a gift from Gates Ventures (D.D.S., D.B.), the Audacious Project at the Institute for Protein Design (H.L.H., H.C., J.D., H.N., D.B.), a gift from Amgen (M.A., D.B.), a grant from DARPA supporting the Harnessing Enzymatic Activity for Lifesaving Remedies (HEALR) program (HR001120S0052 contract HR0011-21-2-0012, X.L., A.K.B., D.B.), an ERC DiProPhys grant from the European Research Council under the Horizon 2020 research and innovation program (agreement ID 101001615, T.P.J.K.), a Canadian Institutes of Health research grant (FND-503573, L.E.K.) and Natural Sciences and Engineering Research Council of Canada grant (2015-04347, L.E.K.). Crystallographic data were collected at the Advanced Photon Source (APS) Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357.Segments of proteins with high β-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in β-strand and β-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein-peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid β1-42 (Aβ42). The Aβ binders block the assembly of Aβ fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aβ42 species