Cortina George A, Hays Jennifer M, Kasson Peter M
Department of Molecular Physiology and Biological Physics, University of Virginia, Box 800886 Charlottesville VA 22908.
Department of Biomedical Engineering, University of Virginia, Box 800886 Charlottesville VA 22908.
ACS Catal. 2018 Apr 6;8(4):2741-2747. doi: 10.1021/acscatal.7b03832. Epub 2018 Feb 21.
The KPC-2 carbapenemase enzyme is responsible for drug resistance in the majority of carbapenem-resistant gram-negative bacterial infections in the United States. A better understanding of what permits KPC-2 to hydrolyze carbapenem antibiotics and how this might be inhibited is thus of fundamental interest and great practical importance to development of better anti-infectives. By correlating molecular dynamics simulations with experimental enzyme kinetics, we have identified conformational changes that control KPC-2's ability to hydrolyze carbapenem antibiotics. Related beta-lactamase enzymes can interconvert between catalytically permissive and catalytically nonpermissive forms of an acylenzyme intermediate critical to drug hydrolysis. Using molecular dynamics simulations, we identify a similar equilibrium in KPC-2 and analyze the determinants of this conformational change. Because the conformational dynamics of KPC-2 are complex and sensitive to allosteric changes, we develop an information-theoretic approach to identify key determinants of this change. We measure unbiased estimators of the reaction coordinate between catalytically permissive and nonpermissive states, perform information-theoretic feature selection and, using restrained molecular dynamics simulations, validate the protein conformational changes predicted to control catalytically permissive geometry. We identify two binding-pocket residues that control the conformational transitions between catalytically active and inactive forms of KPC-2. Mutations to one of these residues, Trp105, lower the stability of the catalytically permissive state in simulations and have reduced experimental values that show a strong linear correlation with the simulated catalytically permissive state lifetimes. This understanding can be leveraged to predict the drug resistance of further KPC-2 mutants and help design inhibitors to combat extreme drug resistance.
KPC-2碳青霉烯酶是美国大多数耐碳青霉烯革兰氏阴性菌感染产生耐药性的原因。因此，更好地了解KPC-2能够水解碳青霉烯抗生素的机制以及如何抑制这种机制，对于开发更好的抗感染药物具有根本的重要意义和巨大的实际重要性。通过将分子动力学模拟与实验酶动力学相关联，我们确定了控制KPC-2水解碳青霉烯抗生素能力的构象变化。相关的β-内酰胺酶可以在对药物水解至关重要的酰基酶中间体的催化允许形式和催化不允许形式之间相互转换。利用分子动力学模拟，我们在KPC-2中确定了类似的平衡，并分析了这种构象变化的决定因素。由于KPC-2的构象动力学复杂且对变构变化敏感，我们开发了一种信息论方法来确定这种变化的关键决定因素。我们测量催化允许状态和非允许状态之间反应坐标的无偏估计量，进行信息论特征选择，并使用受限分子动力学模拟验证预测控制催化允许几何形状的蛋白质构象变化。我们确定了两个控制KPC-2催化活性和非活性形式之间构象转变的结合口袋残基。对其中一个残基色氨酸105进行突变，在模拟中降低了催化允许状态的稳定性，并降低了实验值，这些值与模拟的催化允许状态寿命呈现出强烈的线性相关性。这种认识可用于预测更多KPC-2突变体的耐药性，并有助于设计抑制剂以对抗极端耐药性。
