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Mutagenesis and structural analysis reveal the CTX-M β-lactamase active site is optimized for cephalosporin catalysis and drug resistance
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Toward Guided Mutagenesis: Gaussian Process Regression Predicts MHC Class II Antigen Mutant Binding
Antigen-specific immunotherapies (ASI) require successful loading and presentation of antigen peptides into the major histocompatibility complex (MHC) binding cleft. One route of ASI design is to mutate native antigens for either stronger or weaker binding interaction to MHC. Exploring all possible mutations is costly both experimentally and computationally. To reduce experimental and computational expense, here we investigate the minimal amount of prior data required to accurately predict the relative binding affinity of point mutations for peptide-MHC class II (pMHCII) binding. Using data from different residue subsets, we interpolate pMHCII mutant binding affinities by Gaussian process (GP) regression of residue volume and hydrophobicity. We apply GP regression to an experimental data set from the Immune Epitope Database, and theoretical data sets from NetMHCIIpan and Free Energy Perturbation calculations. We find that GP regression can predict binding affinities of nine neutral residues from a six-residue subset with an average R 2 coefficient of determination value of 0.62 ± 0.04 (±95% CI), average error of 0.09 ± 0.01 kcal/mol (±95% CI), and with an receiver operating characteristic (ROC) AUC value of 0.92 for binary classification of enhanced or diminished binding affinity. Similarly, metrics increase to an R2 value of 0.69 ± 0.04, average error of 0.07 ± 0.01 kcal/mol, and an ROC AUC value of 0.94 for predicting seven neutral residues from an eight-residue subset. Our work finds that prediction is most accurate for neutral residues at anchor residue sites without register shift. This work holds relevance to predicting pMHCII binding and accelerating ASI design.
Combining Reactive Quantum-Mechanical Molecular-Dynamics Simulations with Mutagenesis, Crystallography, and Enzyme Kinetics to Reveal Plausible Steps of Isocyanide Hydratase Catalysis
A complete understanding of enzyme mechanisms requires atomistic details of chemical reactions. Quantum-based molecular dynamics simulations (QMD) are a potential source of this information, but trade-offs between accuracy and computational cost have limited their use. We previously developed extended Lagrangian Born–Oppenheimer molecular dynamics (XL-BOMD) methods that leverage a negligible compromise in accuracy to substantially decrease the cost of QMD simulations. Here, we develop a reactive QMD approach using the latest XL-BOMD formulation, which enables efficient simulations of highly reactive systems, and use it to investigate mechanisms of intermediate formation in isocyanide hydratase (ICH) catalysis. In QMD simulations, molecular analogs of ICH active site residues reacted with para-nitrophenyl isocyanide, forming a thioimidate. Analysis of simulated atomic configurational and charge dynamics revealed a pathway where protonation of the isocyanide carbon occurs prior to thioimidate formation and suggested a possible role of Asp17 as a proton donor in the early phase of ICH catalysis. To test whether the pathway seen using the reactive QMD approach might be relevant to ICH catalysis, we performed X-ray crystallography and pre-steady-state enzyme kinetics studies of wild-type and D17N mutant ICH. Both the structure and kinetics are sensitive to the D17N mutation in a manner that is consistent with the order of the reaction steps seen in the simulations. Mobile protons play essential roles in many enzymes, yet they are difficult to observe experimentally, making the ordering of proton-dependent steps ambiguous in many enzyme mechanisms. The ability to directly simulate model reactions for the design of experiments that provide information about enzyme mechanisms involving mobile protons demonstrates the significance of our reactive QMD approach and motivates further biological applications.
Targeted mutagenesis of the herpesvirus fusogen central helix captures transition states
Herpesviruses remain a burden for animal and human health, including the medically important varicella-zoster virus (VZV). Membrane fusion mediated by conserved core glycoproteins, the fusogen gB and the heterodimer gH-gL, enables herpesvirus cell entry. The ectodomain of gB orthologs has five domains and is proposed to transition from a prefusion to postfusion conformation but the functional relevance of the domains for this transition remains poorly defined. Here we describe structure-function studies of the VZV gB DIII central helix targeting residues 526 EHV 528 . Critically, a H527P mutation captures gB in a prefusion conformation as determined by cryo-EM, a loss of membrane fusion in a virus free assay, and failure of recombinant VZV to spread in cell monolayers. Importantly, two predominant cryo-EM structures of gB[H527P] are identified by 3D classification and focused refinement, suggesting they represented gB conformations in transition. These studies reveal gB DIII as a critical element for herpesvirus gB fusion function.
Combining random mutagenesis, structure-guided design and next-generation sequencing to mitigate pol
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Flipping the substrate preference of Hazara virus ovarian tumour domain protease through structure-based mutagenesis
Abstract not provided
Mutagenesis of Human Cytomegalovirus Glycoprotein L Disproportionately Disrupts gH/gL/gO over gH/gL/pUL128-131
Herein, cell-free and cell-to-cell spread of herpesviruses involves a core fusion apparatus comprised of the fusion protein glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL/gO and gH/gL/pUL128-131 facilitate spread in different cell types. The gO and pUL128-131 components bind distinct receptors, but how the gH/gL portions of the complexes functionally compare is not understood. We previously characterized a panel of gL mutants by transient expression and showed that many were impaired for gH/gL-gB-dependent cell-cell fusion but were still able to form gH/gL/pUL128-131 and induce receptor interference. Here, the gL mutants were engineered into the HCMV BAC clones TB40/e-BAC4 (TB), TR, and Merlin (ME), which differ in their utilization of the two complexes for entry and spread. Several of the gL mutations disproportionately impacted gH/gL/gO-dependent entry and spread over gH/gL/pUL128-131 processes. The effects of some mutants could be explained by impaired gH/gL/gO assembly, but other mutants impacted gH/gL/gO function. Soluble gH/gL/gO containing the L201 mutant failed to block HCMV infection despite unimpaired binding to PDGFRα, indicating the existence of other important gH/gL/gO receptors. Another mutant (L139) enhanced the gH/gL/gO-dependent cell-free spread of TR, suggesting a “hyperactive” gH/gL/gO. Recently published crystallography and cryo-electron microscopy studies suggest structural conservation of the gH/gL underlying gH/gL/gO and gH/gL/pUL128-131. However, our data suggest important differences in the gH/gL of the two complexes and support a model in which gH/gL/gO can provide an activation signal for gB.
Charged particle mutagenesis at low dose and fluence in mouse splenic T cells
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Radiation effects in Caenorhabditis elegans - Mutagenesis by high and low LET ionizing radiation
The nematode C. elegans was used to measure the effectiveness of high-energy ionized particles in the induction of three types of genetic lesions. Recessive lethal mutations in a 40-map unit autosomal region, sterility, and X-chromosome nondisjunction or damage were investigated. Induction rates were measured as a function of linear energy transfer, LET(infinity), for nine ions of atomic nunmber 1-57 accelerated at the BEVALAC accelerator. Linear kinetics were observed for all three types of lesions within the dose/fluence ranges tested and were found to vary strongly as a function of particle LET(infinity). Relative biological effectiveness (RBE) values of up to 4.2 were measured, and action cross sections were calculated and compared to mutagenic responses in other systems.
Katz model prediction of Caenorhabditis elegans mutagenesis on STS-42
Response parameters that describe the production of recessive lethal mutations in C. elegans from ionizing radiation are obtained with the Katz track structure model. The authors used models of the space radiation environment and radiation transport to predict and discuss mutation rates for C. elegans on the IML-1 experiment aboard STS-42.
Estimates of cellular mutagenesis from cosmic rays
A parametric track structure model is used to estimate the cross section as a function of particle velocity and charge for mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in human fibroblast cell cultures. Experiments that report the fraction of mutations per surviving cell for human lung and skin fibroblast cells indicate small differences in the mutation cross section for these two cell lines when differences in inactivation rates between these cell lines are considered. Using models of cosmic ray transport, the mutation rate at the HGPRT locus is estimated for cell cultures in space flight and rates of about 2 to 10 x 10(exp -6) per year are found for typical spacecraft shielding. A discussion of how model assumptions may alter the predictions is also presented.
Charged-particle mutagenesis 2. Mutagenic effects of high energy charged particles in normal human fibroblasts
The biological effects of high Linear Energy Transfer (LET) charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 sq micrometer and 0.09 to 5.56 x 10(exp -3) sq micrometer respectively. The maximum values were obtained by Fe-56 with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(exp -5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.
Mutagenesis in human cells with accelerated H and Fe ions
The overall goals of this research were to determine the risks of mutation induction and the spectra of mutations induced by energetic protons and iron ions at two loci in human lymphoid cells. During the three year grant period the research has focused in three major areas: (1) the acquisition of sufficient statistics for human TK6 cell mutation experiments using Fe ions (400 MeV/amu), Fe ions (600 MeV/amu) and protons (250 MeV/amu); (2) collection of thymidine kinase- deficient (tk) mutants or hypoxanthine phosphoribosyltransferase deficient (hprt) mutants induced by either Fe 400 MeV/amu, Fe 600 MeV/amu, or H 250 MeV/amu for subsequent molecular analysis; and (3) molecular characterization of mutants isolated after exposure to Fe ions (600 MeV/amu). As a result of the shutdown of the BEVALAC heavy ion accelerator in December 1992, efforts were rearranged somewhat in time to complete our dose-response studies and to complete mutant collections in particular for the Fe ion beams prior to the shutdown. These goals have been achieved. A major effort was placed on collection, re-screening, and archiving of 3 different series of mutants for the various particle beam exposures: tk-ng mutants, tk-sg mutants, and hprt-deficient mutants. Where possible, groups of mutants were isolated for several particle fluences. Comparative analysis of mutation spectra has occured with characterization of the mutation spectrum for hprt-deficient mutants obtained after exposure of TK6 cells to Fe ions (600 MeV/amu) and a series of spontaneous mutants.
Insertional Mutagenesis for Genes involved in Otic/Vestibular Development and Function in Xenopus Tropicalis
Sensitivity to gravity is essential for spatial orientation. Consequently, the gravity receptor system is one of the phylogenetically oldest sensory systems, and the special adaptations that enhance sensitivity to gravity are highly conserved. The main goal of this project is to use Xenopus (frog) to identify genes expressed during vestibular and auditory development. These studies will lead a better understanding of the molecular mechanisms involved in vestibular and auditory development and function. We are using a gene-trap approach in Xenopus tropicalis with the green fluorescent protein (GFP) gene as the transgene reporter. GFP expression occurs only when the GFP gene is correctly integrated in actively transcribed genes. Using the GFP as a tag we can easily identify and clone the mutated gene. In addition, we can study the function of the mutated gene by analyzing the defects generated by insertion of the GFP transgene. To date we have tissue specific GFP expression in X. tropicalis including expression in ear, neural tube, kidney, muscle, eyes and nose. Our transgenic animals will soon reach maturity so that we can outcross them and analyze their progeny. Our next goal is to isolate RNA from our transgenics and clone the tagged genes using RACE-PCR. Currently we are optimizing the RACE-PCR method using transgenics with crystallin GFP expression.
Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts
The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 micrometer2 and 0.09 to 5.56 x 10(-3) micrometer2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(-5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.
Heavy ion mutagenesis: linear energy transfer effects and genetic linkage
We have characterized a series of 69 independent mutants at the endogenous hprt locus of human TK6 lymphoblasts and over 200 independent S1-deficient mutants of the human x hamster hybrid cell line AL arising spontaneously or following low-fluence exposures to densely ionizing Fe ions (600 MeV/amu, linear energy transfer = 190 keV/microns). We find that large deletions are common. The entire hprt gene (> 44 kb) was missing in 19/39 Fe-induced mutants, while only 2/30 spontaneous mutants lost the entire hprt coding sequence. When the gene of interest (S1 locus = M1C1 gene) is located on a nonessential human chromosome 11, multilocus deletions of several million base pairs are observed frequently. The S1 mutation frequency is more than 50-fold greater than the frequency of hprt mutants in the same cells. Taken together, these results suggest that low-fluence exposures to Fe ions are often cytotoxic due to their ability to create multilocus deletions that may often include the loss of essential genes. In addition, the tumorigenic potential of these HZE heavy ions may be due to the high potential for loss of tumor suppressor genes. The relative insensitivity of the hprt locus to mutation is likely due to tight linkage to a gene that is required for viability.