Browsing by Author "Vos, Matthijn"

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    3D variability analysis reveals a hidden conformational change controlling ammonia transport in human asparagine synthetase
    (Springer Nature, 2024-12-03) Coricello, Adriana; Nardone, Alanya J.; Lupia, Antonio; Gratteri, Carmen; Vos, Matthijn; Chaptal, Vincent; Alcaro, Stefano; Zhu, Wen; Takagi, Yuichiro; Richards, Nigel G. J.; Biochemistry and Molecular Biology, School of Medicine
    Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we show that 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) identifies a functional role for the Arg-142 side chain and test this hypothesis experimentally by characterizing the R142I variant in which Arg-142 is replaced by isoleucine. Support for Arg-142 playing a role in the intramolecular translocation of ammonia between the active site of the enzyme is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS, and MD simulations provide a possible molecular mechanism for these findings. Combining 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.
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    Cryo-EM and Molecular Dynamics Simulations Reveal Hidden Conformational Dynamics Controlling Ammonia Transport in Human Asparagine Synthetase
    (bioRxiv, 2023-05-16) Coricello, Adriana; Zhu, Wen; Lupia, Antonio; Gratteri, Carmen; Vos, Matthijn; Chaptal, Vincent; Alcaro, Stefano; Takagi, Yuichiro; Richards, Nigel G. J.; Biochemistry and Molecular Biology, School of Medicine
    How dynamical motions in enzymes might be linked to catalytic function is of significant general interest, although almost all relevant experimental data, to date, has been obtained for enzymes with a single active site. Recent advances in X-ray crystallography and cryogenic electron microscopy offer the promise of elucidating dynamical motions for proteins that are not amenable to study using solution-phase NMR methods. Here we use 3D variability analysis (3DVA) of an EM structure for human asparagine synthetase (ASNS) in combination with atomistic molecular dynamics (MD) simulations to detail how dynamic motions of a single side chain mediates interconversion of the open and closed forms of a catalytically relevant intramolecular tunnel, thereby regulating catalytic function. Our 3DVA results are consistent with those obtained independently from MD simulations, which further suggest that formation of a key reaction intermediate acts to stabilize the open form of the tunnel in ASNS to permit ammonia translocation and asparagine formation. This conformational selection mechanism for regulating ammonia transfer in human ASNS contrasts sharply with those employed in other glutamine-dependent amidotransferases that possess a homologous glutaminase domain. Our work illustrates the power of cryo-EM to identify localized conformational changes and hence dissect the conformational landscape of large proteins. When combined with MD simulations, 3DVA is a powerful approach to understanding how conformational dynamics regulate function in metabolic enzymes with multiple active sites.