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Glycoside hydrolase enzymes are important for hydrolyzing the β-1,4 glycosidic bond in polysaccharides for deconstruction of carbohydrates. The two-step retaining reaction mechanism of Glycoside Hydrolase Family 7 (GH7) was explored with different sized QM-cluster models built by the Residue Interaction Network ResidUe Selector (RINRUS) software using both the wild-type protein and its E217Q mutant. The first step is the glycosylation, in which the acidic residue 217 donates a proton to the glycosidic oxygen leading to bond cleavage. In the subsequent deglycosylation step, one water molecule migrates into the active site and attacks the anomeric carbon. Residue interaction-based QM-cluster models lead to reliable structural and energetic results for proposed glycoside hydrolase mechanisms. The free energies of activation for glycosylation in the largest QM-cluster models were predicted to be 19.5 and 31.4 kcal mol −1 for the wild-type protein and its E217Q mutant, which agree with experimental trends that mutation of the acidic residue Glu217 to Gln will slow down the reaction; and are higher in free energy than the deglycosylation transition states (13.8 and 25.5 kcal mol −1 for the wild-type protein and its mutant, respectively). For the mutated protein, glycosylation led to a low-energy product. This thermodynamic sink may correspond to the intermediate state which was isolated in the X-ray crystal structure. Hence, the glycosylation is validated to be the rate-limiting step in both the wild-type and mutated enzyme.
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Energetic and structural insights behind calcium induced conformational transition in calmodulin
Abstract Calmodulin (CaM) is a key signaling protein that triggers several cellular and physiological processes inside the cell. Upon binding with calcium ion, CaM undergoes large scale conformational transition from a closed state to an open state that facilitates its interaction with various target protein and regulates their activity. This work explores the origin of the energetic and structural variation of the wild type and mutated CaM and explores the molecular origin for the structural differences between them. We first calculated the sequential calcium binding energy to CaM using the PDLD/S‐LRA/β approach. This study shows a very good correlation with experimental calcium binding energies. Next we calculated the calcium binding energies to the wild type CaM and several mutated CaM systems which were reported experimentally. On the structural aspect, it has been reported experimentally that certain mutation (Q41L‐K75I) in calcium bound CaM leads to complete conformational transition from an open to a closed state. By using equilibrium molecular dynamics simulation, free energy calculation and contact frequency map analysis, we have shown that the formation of a cluster of long‐range hydrophobic contacts, initiated by the Q41L‐K75I CaM variant is the driving force behind its closing motion. This study unravels the energetics and structural aspects behind calcium ion induced conformational changes in wild type CaM and its variant.
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- PAR ID:
- 10489838
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Proteins: Structure, Function, and Bioinformatics
- Volume:
- 92
- Issue:
- 3
- ISSN:
- 0887-3585
- Format(s):
- Medium: X Size: p. 384-394
- Size(s):
- p. 384-394
- Sponsoring Org:
- National Science Foundation
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