Side Chain Conformational Averaging in Human Dihydrofolate Reductase

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• 作者：Lisa M. Tuttle ; H. Jane Dyson ; Peter E. Wright
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：February 25, 2014
• 年：2014
• 卷：53
• 期：7
• 页码：1134-1145
• 全文大小：921K
• 年卷期：v.53,no.7(February 25, 2014)
• ISSN：1520-4995

文摘

The three-dimensional structures of the dihydrofolate reductase enzymes from Escherichia coli (ecDHFR or ecE) and Homo sapiens (hDHFR or hE) are very similar, despite a rather low level of sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and E. coli enzymes, we conducted a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations cannot be detected for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that helix 伪F changes position by sliding between the two states. Analysis of 蠂₁ rotamer populations derived from measurements of ³J_C纬CO and ³J_C纬N couplings indicates that many of the side chains that contact helix 伪F exhibit rotamer averaging that may facilitate the conformational change. The 蠂₁ rotamer adopted by the Phe31 side chain depends upon whether the active site contains the substrate or product. In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 蠂₁ rotamer opens the active site to facilitate entry of the substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as it proceeds through its enzymatic cycle, but that ligand flux is brokered by more subtle conformational changes that depend on the side chain motions of critical residues.