Abstract
Proteins, especially multi-domain proteins, often undergo drastic conformational changes upon binding to ligands or by post-translational modifications, which is a key step to regulate their function. However, the detailed mechanisms of such dynamic regulation of the functional processes are poorly understood because of the lack of an efficient tool. We here demonstrate detailed characterization of conformational changes of MurD, a 47 kDa protein enzyme consisting of three domains, by the use of solution NMR equipped with paramagnetic lanthanide probe. Quantitative analysis of pseudocontact shifts has identified a novel conformational state of MurD, named semi-closed conformation, which is found to be the key to understand how MurD regulates the binding of the ligands. The modulation of the affinity coupled with conformational changes accentuates the importance of conformational state to be evaluated in drug design.
Highlights
To investigate the dynamic structural changes of domain 3 coupled with the enzymatic process, we have exploited paramagnetic lanthanide probe method[6], where paramagnetic lanthanide ion is fixed in a protein frame by the use of a lanthanide-binding tag[12,13,14,15] and long-range structural information is extracted from paramagnetic effects such as pseudocontact shift (PCS)
Combined with nuclear magnetic resonance (NMR) titration and isothermal titration calorimetry, the PCS data show that the ligand binding triggers conformational change of MurD that modulates the affinity to the subsequent ligand to regulate the order of the ligand binding
We investigated the affinities between MurD and its ligands by NMR and isothermal titration calorimetry (ITC)
Summary
Further addition of the ligands didn’t change the PCSs, proving that the binding was saturated (data not shown) These observations implied the existence of another conformational state of MurD that has been veiled in the previous crystallographic studies. Different PCS profiles were observed in the presence of UMA: Addition of UMA didn’t cause further PCS change in the presence of AMP-PNP and Mg2+, while addition of UMA in the presence of ADP and Mg2+ induced further decrease in the magnitude of PCSs, resulting in the PCSs very close to the theoretical values for closed state (Fig. 3a,b,e) This observation implied that the hydrolysis of ATP triggers further conformational change from semi-closed to fully closed state.
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