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Computational Research in Molecular Chemistry
Integrating comparative modeling and accelerated simulations reveals conformational and energetic basis of actomyosin force generation.Ma, Y., S. You, M. Regnier, J.A. McCammonProc. Natl. Acad. Sci. USA (2023) 120 (9) e2215836120    
Muscle contraction is performed by arrays of contractile proteins in the sarcomere. Serious heart diseases, such as cardiomyopathy, can often be results of mutations in myosin and actin. Direct characterization of how small changes in the myosin-actin complex impact its force production remains challenging. Molecular dynamics (MD) simulations, although capable of studying protein structurefunction relationships, are limited owing to the slow timescale of the myosin cycle as well as a lack of various intermediate structures for the actomyosin complex. Here, employing comparative modeling and enhanced sampling MD simulations, we show how the human cardiac myosin generates force during the mechanochemical cycle. Initial conformational ensembles for different myosin-actin states are learned from multiple structural templates with Rosetta. This enables us to efficiently sample the energy landscape of the system using Gaussian accelerated MD. Key myosin loop residues, whose substitutions are related to cardiomyopathy, are identified to form stable or metastable interactions with the actin surface. We find that the actin-binding cleft closure is allosterically coupled to the myosin core transitions and ATP-hydrolysis product release from the active site. Furthermore, a gate between switch I and switch II is suggested to control phosphate release at the pre-powerstroke state. Our approach demonstrates the ability to link sequence and structural information to motor functions.
Editorial: Mechanisms, thermodynamics and kinetics of ligand binding revealed from molecular simulations and machine learningMiao, Y., C.A. Chang, W. Zhu, J.A. McCammonFront. Molec.Bioscience (2023) 17 January 2023, Sec. Molecular Recognition Volume 10 - 2023    
PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.Philpott, J.M., A.M. Freeberg, J. Park, K. Lee, C.G. Ricci, S.R. Hunt, R. Narasimamurthy, D.H. Segal, R. Robles, Y. Cai, S. Tripathi, J.A. McCammon, D.M. Virshup, J.C. Chiu, C. Lee, C.L. Partch.Molecular Cell, (2023), Vol. 83, Issue 10, p1677-1692, e8 May 18, 2023    
PERIOD (PER) and Casein Kinase 1δ regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1δ phosphorylation of the familial advanced sleep phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortening circadian period in human cells. We found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.
Capturing differences in the regulation of LRRK2 dynamics and conformational states by small molecule kinase inhibitors.Weng, J.-H., W. Ma, J. Wu, P. Kaila Sharma, S. Silletti, J.A. McCammon, S. Taylor.ACS Chem. Biol. 2023, 18, 4, 810–821    
Mutations in the human leucine rich repeat protein kinase-2 (LRRK2) create risk factors for Parkinson’s disease, and pathological functions of LRRK2 are often correlated with aberrant kinase activity. Past research has focused on developing selective LRRK2 kinase inhibitors. In this study, we combined enhanced sampling simulations with HDX-MS to characterize the inhibitor-induced dynamic changes and the allosteric communications within the C-terminal domains of LRRK2, LRRK2RCKW. We find that the binding of MLi-2 (a type I kinase inhibitor) stabilizes a closed kinase conformation and reduces the global dynamics of LRRK2RCKW, leading to a more compact LRRK2RCKW structure. In contrast, the binding of Rebastinib (a type II kinase inhibitor) stabilizes an open kinase conformation, which promotes a more extended LRRK2RCKW structure. By probing the distinct effects of the type I and type II inhibitors, key interdomain interactions are found to regulate the communication between the kinase domain and the GTPase domain. The intermediate states revealed in our simulations facilitate the efforts toward in silico design of allosteric modulators that control LRRK2 conformations and potentially mediate the oligomeric states of LRRK2 and its interactions with other proteins.
Multiscale Computational Modeling of the Effects of 2-deoxy-ATP on Cardiac Muscle Calcium HandlingHock, M.T., A.E. Teitgen, K.J. McCabe, S.P. Hirakis, G.A. Huber, M. Regnier, R.E. Amaro, J.A. McCammon, A.D. McCulloch.J. Appl. Phys. 134, 074905 (2023)    
2'-Deoxy-ATP (dATP), a naturally occurring near analog of ATP, is a well-documented myosin activator that has been shown to increase contractile force, improve pump function, and enhance lusitropy in the heart. Calcium transients in cardiomyocytes with elevated levels of dATP show faster calcium decay compared with cardiomyocytes with basal levels of dATP, but the mechanisms behind this are unknown. Here, we design and utilize a multiscale computational modeling framework to test the hypothesis that dATP acts on the sarcoendoplasmic reticulum calcium-ATPase (SERCA) pump to accelerate calcium re-uptake into the sarcoplasmic reticulum during cardiac relaxation. Gaussian accelerated molecular dynamics simulations of human cardiac SERCA2A in the E1 apo, ATP-bound and dATP-bound states showed that dATP forms more stable contacts in the nucleotide binding pocket of SERCA and leads to increased closure of cytosolic domains. These structural changes ultimately lead to changes in calcium binding, which we assessed using Brownian dynamics simulations. We found that dATP increases calcium association rate constants to SERCA and that dATP binds to apo SERCA more rapidly than ATP. Using a compartmental ordinary differential equation model of human cardiomyocyte excitation-contraction coupling, we found that these increased association rate constants contributed to the accelerated rates of calcium transient decay observed experimentally. This study provides clear mechanistic evidence of enhancements in cardiac SERCA2A pump function due to interactions with dATP.
Interface Engineering of Carrier Protein–Dependent Metabolic PathwaySztain, T., J.C. Corpuz, T. Bartholow, J. Sanlley, Z. Jiang, D. Mellor, G.W. Heberling, J. LaClair, J.A. McCammon, M.D. BurkartACS Chem. Biol. 2023, 18, 9, 2014–2022 Publication Date:September 6, 2023    
Carrier-protein-dependent metabolic pathways biosynthesize fatty acids, polyketides, and non-ribosomal peptides, producing metabolites with important pharmaceutical, environmental, and industrial properties. Recent findings demonstrate that these pathways rely on selective communication mechanisms involving protein–protein interactions (PPIs) that guide enzyme reactivity and timing. While rational design of these PPIs could enable pathway design and modification, this goal remains a challenge due to the complex nature of protein interfaces. Computational methods offer an encouraging avenue, though many score functions fail to predict experimental observables, leading to low success rates. Here, we improve upon the Rosetta score function, leveraging experimental data through iterative rounds of computational prediction and mutagenesis, to design a hybrid fatty acid–non-ribosomal peptide initiation pathway. By increasing the weight of the electrostatic score term, the computational protocol proved to be more predictive, requiring fewer rounds of iteration to identify mutants with high in vitro activity. This allowed efficient design of new PPIs between a non-ribosomal peptide synthetase adenylation domain, PltF, and a fatty acid synthase acyl carrier protein, AcpP, as validated by activity and structural studies. This method provides a promising platform for customized pathway design, establishing a standard for carrier-protein-dependent pathway engineering through PPI optimization.
Glycosylation and Crowded Membrane Effects on Influenza Neuraminidase Stability and Dynamics.Seitz C., I. Deveci, J.A. McCammonJ Phys Chem Lett. 14, 9926-9934 (2023).    
All protein simulations are conducted with varying degrees of simplification, oftentimes with unknown ramifications about how these simplifications affect the interpretability of the results. In this work, we investigated how protein glycosylation and lateral crowding effects modulate an array of properties characterizing the stability and dynamics of influenza neuraminidase. We constructed three systems: (1) glycosylated neuraminidase in a whole virion (i.e., crowded membrane) environment, (2) glycosylated neuraminidase in its own lipid bilayer, and (3) unglycosylated neuraminidase in its own lipid bilayer. We saw that glycans tend to stabilize the protein structure and reduce its conformational flexibility while restricting the solvent movement. Conversely, a crowded membrane environment encouraged exploration of the free energy landscape and a large-scale conformational change, while making the protein structure more compact. Understanding these effects informs what factors one must consider in attempting to recapture the desired level of physical accuracy.
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