Characterization of the Domain Orientations of E. coli 5′-Nucleotidase by Fitting an Ensemble of Conformers to DEER Distance Distributions

• Published in: Structure (London) 2016-01, Vol.24 (1), p.43-56
• Main Authors: Krug, Ulrike, Alexander, Nathan S., Stein, Richard A., Keim, Antje, Mchaourab, Hassane S., Sträter, Norbert, Meiler, Jens
• Format: Article
• Language: English
• Subjects: 5'-Nucleotidase - chemistry; 5'-Nucleotidase - metabolism; Amino Acid Sequence; Escherichia coli - enzymology; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Molecular Dynamics Simulation; Molecular Sequence Data; Protein Structure, Tertiary
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2015.11.007

Escherichia coli 5′-nucleotidase is a two-domain enzyme exhibiting a unique 96° domain motion that is required for catalysis. Here we present an integrated structural biology study that combines DEER distance distributions with structural information from X-ray crystallography and computational biology to describe the population of presumably almost isoenergetic open and closed states in solution. Ensembles of models that best represent the experimental distance distributions are determined by a Monte Carlo search algorithm. As a result, predominantly open conformations are observed in the unliganded state indicating that the majority of enzyme molecules await substrate binding for the catalytic cycle. The addition of a substrate analog yields ensembles with an almost equal mixture of open and closed states. Thus, in the presence of substrate, efficient catalysis is provided by the simultaneous appearance of open conformers (binding substrate or releasing product) and closed conformers (enabling the turnover of the substrate). [Display omitted] •Almost isoenergetic states of a domain motion can be characterized by EPR•X-Ray structures are combined with spectroscopic data by ensemble fitting•A quantitative characterization of the equilibrium can be obtained•Substrate binding changes the equilibrium from open to open and closed states Krug et al. combine EPR distance distributions, information from crystal structures and an ensemble fitting procedure by a Monte Carlo search algorithm in an integrated study to describe the conformational population of E. coli 5′-nucleotidase in the apo- and inhibitor-bound states.