McCammon Group
NewsSeminarsPublicationsPeopleWikiContact Us
McCammon Group
We have recently changed our website. Looking for the previous site?
Computational Research in Molecular Chemistry
Activation of atypical protein kinase C by sphingosine 1-phosphate revealed by an aPKC-specific activity reporterKajimoto, T., A.D. Caliman, I.S. Tobias, T. Okada, C.A. Pilo, A. Van, J.A. McCammon, S. Nakamura, A.C. NewtonScience Signal., Volume 12, Issue 562, eaat6662 (2019).    
Atypical protein kinase C (aPKC) isozymes are unique in the PKC superfamily in that they are not regulated by the lipid second messenger diacylglycerol, which has led to speculation about whether a different second messenger acutely controls their function. Here, using a genetically encoded reporter that we designed, aPKC-specific C kinase activity reporter (aCKAR), we found that the lipid mediator sphingosine 1-phosphate (S1P) promoted the cellular activity of aPKC. Intracellular S1P directly bound to the purified kinase domain of aPKC and relieved autoinhibitory constraints, thereby activating the kinase. In silico studies identified potential binding sites on the kinase domain, one of which was validated biochemically. In HeLa cells, S1P-dependent activation of aPKC suppressed apoptosis. Together, our findings identify a previously undescribed molecular mechanism of aPKC regulation, a molecular target for S1P in cell survival regulation, and a tool to further explore the biochemical and biological functions of aPKC.
Docking simulation and antibiotic discovery targeting the MlaC protein in Gram-negative bacteriaHuang, Y.-M., J. Munguia, Y. Miao, V. Nizet, J.A. McCammonChemBiol. Drug Disc., Vol. 93 (4) pp 647-658 (2019)    
To maintain the lipid asymmetry of the cell envelope in Gram-negative bacteria, the MlaC protein serves as a lipid-transfer factor and delivers phospholipids from the outer to the inner membrane. A strategy of antibiotic discovery is to design a proper compound that can tightly bind to the MlaC protein and inhibit the MlaC function. In this study, we performed virtual screening on multiple MlaC structures obtained from molecular dynamics simulations to identify potential MlaC binders. Our results suggested that clorobiocin is a compound that could bind to the MlaC protein. Through the comparison of the bound geometry between clorobiocin and novobiocin, we pointed out that the methyl-pyrrole group of the noviose sugar in clorobiocin forms hydrophobic interactions with amino acids in the phospholipid binding pocket, which allows the compound to bind deep in the active site. This also explains why clorobiocin shows a tighter binding affinity than novobiocin. Our study highlights a practical path of antibiotic development against Gram-negative bacteria.
pH dependent conformational dynamics of Beta-secretase 1: A molecular dynamics studyMermelstein, D., J.A. McCammon, R. Walker.J. Mol. Recog. 32, e2765 (2019).    
Beta-secretase 1 (BACE-1) is an aspartyl protease implicated in the overproduction of β-amyloid fibrils responsible for Alzheimer’s disease. The process of β-amyloid genesis is known to be pH dependent, with an activity peak between solution pH of 3.5 and 5.5. We have studied the pH dependent dynamics of BACE-1 to better understand the pH dependent mechanism. We have implemented support for graphics processor unit (GPU) accelerated constant pH molecular dynamics within the AMBER molecular dynamics software package and employed this to determine the relative population of different aspartyl dyad protonation states in the pH range of greatest β-amyloid production, followed by conventional molecular dynamics to explore the differences among the various Aspartyl dyad protonation states. We observed a difference in dynamics between double protonated, mono-protonated, and double deprotonated states over the known pH range of higher activity. These differences include Tyr 71-Aspartyl dyad proximity and active water lifetime. This work indicates that Tyr 71 stabilizes catalytic water in the Aspartyl dyad active site, enabling BACE-1 activity.
Mechanistic Probes for the Epimerization Domain of Nonribosomal Peptide SynthetasesKim, W.E., A. Patel, G.H. Hur, P. Tufar, M.G. Wuo, J.A. McCammon, M.D. BurkartChemBioChem,Vol. 20, Issue 2, pp 1247-152 (2019)    
Nonribosomal peptide synthetases (NRPSs) are responsible for the synthesis of a variety of bioactive natural products with clinical and economic significance. Interestingly, these large multimodular enzyme machineries incorporate nonproteinogenic D-amino acids through the use of auxiliary epimerization domains, converting L-amino acids into D-amino acids, which impart into the resulting natural products unique bioactivity and resistance to proteases. Due to the large and complex nature of NRPSs, several questions remain unanswered about the mechanism of the catalytic domain reactions. Herein, we investigate the use ofmechanism-based crosslinkers to probe the mechanism of an epimerization domain in gramicidin S biosynthesis. In addition, MD simulations were performed, showcasing the possible roles of catalytic residues within the epimerization domain.
Mechanisms for Benzene Dissociation through the Excited State of T4 Lysozyme L99AFeher, V., J. Schiffer, D. Mermelstein, N. Mih, L. Pierce, J.A. McCammon, R. AmaroBiophys. J. Vol. 116, Issue 2, pp 205-214 (2019).    
The atomic-level mechanisms that coordinate ligand release from protein pockets are only known for a handful of proteins. Here, we report results from accelerated molecular dynamics simulations for benzene dissociation from the buried cavity of the T4 lysozyme Leu99Ala mutant (L99A). In these simulations, benzene is released through a previously characterized, sparsely populated room-temperature excited state of the mutant, explaining the coincidence for experimentally measured benzene off rate and apo protein slow-timescale NMR relaxation rates between ground and excited states. The path observed for benzene egress is a multistep ligand migration from the buried cavity to ultimate release through an opening between the F/G-, H-, and I-helices and requires a number of cooperative multiresidue and secondary-structure rearrangements within the C-terminal domain of L99A. These rearrangements are identical to those observed along the ground state to excited state transitions characterized by molecular dynamic simulations run on the Anton supercomputer. Analyses of the molecular properties of the residues lining the egress path suggest that protein surface electrostatic potential may play a role in the release mechanism. Simulations of wild-type T4 lysozyme also reveal that benzene-egress-associated dynamics in the L99A mutant are potentially exaggerations of the substrate-processivity-related dynamics of the wild type.
Allostery in its many disguises: from theory to applicationsWodak, S.J., E. Paci, N.V. Dokholyan, I.N. Berezovsky, A. Horovitz, J. Li, V.J. Hilser, I. Bahar, J. Karanicolas, G. Stock, P. Hamm, R.H. Stote, J. Eberhardt, Y. Chebaro, A. Dejaegere, M. Cecchini, J.P. Changeux, D. Keri, P. Barth, P.G. Bolhuis, J. Vreede, P. Faccioli, S. Orioli, R. Ravasio, L. Yan, C. Brito, M. Wyart, P. Gkeka, I. Rivalta, G. Palermo, J.A. McCammon, J. Panecka-Hofman, R.C. Wade, A. Di Pizio, M.Y. Niv, R. Nussinov, C.-J. Tsai, H. Jang, D. Padhorny, D. Kozakov, T. McLeishStructure, Vol. 27, Issue 4, pp 566-578 (2019)    
Structural and dynamical rationale for fatty acid unsaturation in E. coliDodge, G.J., A. Patel, K.L. Jaremko, J.A. McCammon, J.L. Smith, M.D. Burkart.Proc. Natl. Acad. Sci. USA, 116 (14) pp 6775-6783 (2019)    
Fatty acid biosynthesis in α- and γ-proteobacteria requires two functionally distinct dehydratases (DHs), FabA and FabZ. Here, mechanistic crosslinking facilitates the first structural characterization of a stable hexameric complex of six E. coli FabZ dehydratase subunits with six AcpP acyl carrier proteins (ACP) . The crystal structure sheds light on the divergent substrate selectivity of FabA and FabZ by revealing distinct architectures of the binding pocket. Molecular dynamics simulations demonstrate differential biasing of substrate orientations and conformations within the active sites of FabA and FabZ such that FabZ is pre-organized to catalyze only dehydration, while FabA is primed for both dehydration and isomerization.
Deciphering Off-target Effects in CRISPR-Cas9 through Accelerated Molecular DynamicsRicci, C.G., J.S. Chen, Y. Miao, M. Jinik, J.A. Doudna, J.A. McCammon, G. PalermoACS Central Science, 5, 4, pp 651-662 (2019)    
CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations – using Gaussian accelerated MD (GaMD) – are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at distal sites with respect to the Protospacer Adjacent Motif (PAM) can induce an extended opening of the RNA:DNA heteroduplex, which leads to newly formed interactions between the unwound DNA and the L2 loop of the catalytic HNH domain. These conserved interactions constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and hampering its activation for cleavage. Remarkably, depending on their positions at PAM distal sites, DNA mismatches responsible for off-target cleavages are unable to “lock” the HNH domain, thereby leading to the unselective cleavage of DNA sequences. In consistency with the available experimental data, the ability to “lock” the catalytic HNH domain in an inactive “conformational checkpoint” is shown to be a key determinant in the onset of off-target effects. This mechanistic rationale contributes in clarifying a long lasting open issue in the CRISPR-Cas9 function and poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.
The invisible dance of CRISPR-Cas9Palermo, G., C. Ricci, J.A. McCammonPhysics Today, Vol. 72, (4) 30 (2019)    
Understanding the mechanistic basis of non-coding RNA through molecular dynamics simulationsPalermo, G., L. Casalino, A. Magistrato, J.A. McCammonJ. Struct. Biol., Vol. 206 (3) pp 267-279 (2019)    
Noncoding RNA (ncRNA) has a key role in regulating gene expression, mediating fundamental processes and diseases via a variety of yet unknown mechanisms. Here, we review recent applications of conventional and enhanced Molecular Dynamics (MD) simulations methods to address the mechanistic function of large biomolecular systems that are tightly involved in the ncRNA function and that are of key importance in life sciences. This compendium focuses of three biomolecular systems, namely the CRISPR-Cas9 genome editing machinery, group II intron ribozyme and the ribonucleoprotein complex of the spliceosome, which edit and process ncRNA. We show how the application of a novel accelerated MD simulations method has been key in disclosing the conformational transitions underlying RNA binding in the CRISPR-Cas9 complex, suggesting a mechanism for RNA recruitment and clarifying the conformational changes required for attaining genome editing. As well, we discuss the use of mixed quantum-classical MD simulations in deciphering the catalytic mechanism of RNA splicing as operated by group II intron ribozyme, one of the largest ncRNA structures crystallized so far. Finally, we debate the future challenges and opportunities in the field, discussing the recent application of MD simulations for unraveling the functional biophysics of the spliceosome, a multi-mega Dalton complex of proteins and small nuclear RNAs that performs RNA splicing in humans. This showcase of applications highlights the current talent of MD simulations to dissect atomic-level details of complex biomolecular systems instrumental for the design of finely engineered genome editing machines. As well, this review aims at inspiring future investigations of several other ncRNA regulatory systems, such as micro and small interfering RNAs, which achieve their function and specificity using RNA-based recognition and targeting strategies.
The Implementation of the Colored Abstract Simplicial Complex and its Application to Mesh GenerationLee, C.T., J.B. Moody, R.E. Amaro, J.A. McCammon, M.J. Holst.ACM Transactions of Mathematical Software, Vol. 45, (3), Article 28 (2019)    
See code at
One atom matters: modifying the thioester linkage affects structure of the acyl carrier proteinSztain, T., A. Patel, J. Lee, T. Davis, J.A. McCammon, M. Burkart.Angew Chem, 58 (32), pp10888-10892 (2019)    
At the center of many complex biosynthetic pathways, the acyl carrier protein (ACP) shuttles substrates to appropriate enzymatic partners to produce fatty acids and polyketides. Carrier proteins covalently tether their cargo by a thioester linkage to a phosphopantetheine cofactor. Due to the labile nature of this linkage, chemoenzymatic methods have been developed that involve replacement of the thioester with a more stable amide or ester bond. Here we explore the importance of the thioester bond to the structure of the ACP using solution NMR spectroscopy and molecular dynamics simulations. Remarkably, the replacement of sulfur for other heteroatoms results in significant structural changes that can inform the proper selection of isosteric substitutes.
Variational implicit-solvent predictions of the dry-wet transition pathways for ligand-receptor binding and unbinding kineticsZhou, S., R.G. Weiss, L.-T. Cheng, J. Dzubiella, J.A. McCammon, B. Li.Proc. Natl. Acad. Sci. USA , 116 (30), pp14989-14994 (2019)    
Ligand-receptor binding and unbinding are fundamental biomolecular processes and particularly essential to drug efficacy. Environmental water fluctuations, however, impact the corresponding thermodynamics and kinetics and thereby challenge theoretical descriptions. Here, we devise a holistic, implicit-solvent, multi-method approachtopredictthe(un)bindingkineticsforagenericligand-pocket model. We use the variational implicit-solvent model (VISM) to calculate the solute-solvent interfacial structures and the corresponding free energies, and combine the VISM with the string method to obtain the minimum energy paths and transition states between the various metastable (“dry” and “wet”) hydration states. The resulting dry-wet transition rates are then used in a spatially-dependent multi-state continuous-time Markov chain Brownian dynamics simulations, and the related Fokker–Planck equation calculations, of the ligand stochastic motion, providing the mean first-passage times for binding and unbinding. We find the hydration transitions to significantly slow down the binding process, in semi-quantitative agreement with existing explicit-water simulations, but significantly accelerate the unbinding process. Moreover, our methods allow the characterization of non-equilibrium hydration states of pocket and ligand during the ligand movement, for which we find substantial memory and hysteresis effects for binding versus unbinding. Our study thus provides a significant step forward towards efficient, physics-based interpretation and predictions of the complex kinetics in realistic ligand-receptor systems.
Brownian Dynamics Simulations of Biological MoleculesHuber, G., J.A. McCammon.Trends in Chemistry in press (2019).    
Brownian dynamics is a technique for carrying out computer simulations of physical systems that are driven by thermal fluctuations. Biological systems at the macromolecular and cellular level, while falling in the gap between well-established atomic-level models and continuum models, are especially suitable for such simulations. We present a brief history, examples of important biological processes that are driven by thermal motion, and those that have been profitably studied by Brownian dynamics. We also present some of the challenges facing developers of algorithms and software, especially in the attempt to simulate larger systems more accurately and for longer times.
A Coarse-Grained Multiscale Model of Thin Filament Activation using Brownian and Langevin DynamicsAboelkassem, Y., K.J. McCabe, G. Huber, J. Sundnes, M. Regnier, J.A. McCammon, A.D. McCullochBiophys. J. in press (2019)    
Shifting the Hydrolysis Equilibrium of Substrate Loaded Acyl Carrier ProteinsSztain, T., T. Bartholow, J.A. McCammon, M.D. BurkartBiochemistry, 58, 34, pp 3557-3560 (2019)    
Acyl carrier proteins (ACP)s transport intermediates through many primary and secondary metabolic pathways. Studying the effect of substrate identity on ACP structure has been hindered by the lability of the thioester bond that attaches acyl substrates to the 4′-phosphopantetheine cofactor of ACP. Here we show that an acyl acyl-carrier protein synthetase (AasS) can be used in real time to shift the hydrolysis equilibrium toward favoring acyl-ACP during solution NMR spectroscopy. Only 0.005 molar equivalents of AasS enables 1 week of stability to palmitoyl-AcpP from Escherichia coli. 2D NMR spectra enabled with this method revealed that the tethered palmitic acid perturbs nearly every secondary structural region of AcpP. This technique will allow previously unachievable structural studies of unstable acyl-ACP species, contributing to the understanding of these complex biosynthetic pathways.
Website Security Test