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Browsing by Author "Coricello, Adriana"
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Item 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 MedicineHow 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.Item High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity(Springer Nature, 2019-09-17) Zhu, Wen; Radadiya, Ashish; Bisson, Claudine; Wenzel, Sabine; Nordin, Brian E.; Martínez-Márquez, Francisco; Imasaki, Tsuyoshi; Sedelnikova, Svetlana E.; Coricello, Adriana; Baumann, Patrick; Berry, Alexandria H.; Nomanbhoy, Tyzoon K.; Kozarich, John W.; Jin, Yi; Rice, David W.; Takagi, Yuichiro; Richards, Nigel G. J.; Biochemistry and Molecular Biology, School of MedicineExpression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 μM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.