Book of Abstracts: Albany 2011

category image Albany 2011
Conversation 17
June 14-18 2011
©Adenine Press (2010)

Role of Disulfide Bonds in Structural Stability and Flexibility of Cuticle-degrading proteases from nematophagous fungi ⎯ a molecular dynamics simulation study

It has been shown that disulfide bonds play an important role in the stability of some proteins by an entropic effect (1), usually the globular proteins secreted to extracellular medium (2). Two cuticle-degrading proteases, Ver112 and PII, which were derived respectively from nematode-parasitic and nematode-trapping fungi, belong to the subtilisin family sharing relatively high sequence identity (45.7%). Ver112 is an alkaline protease and has two disulfide bonds, C35-C124 and C179-C250 (3); PII is a neutral protease and has no disulfide bond. Despite the minor structural difference between them (root mean square deviation (RMSD) is ~ 0.6 Å), Ver112 displays higher thermal stability and stronger nematicidal/catalytic activity than PII does (4).

In order to investigate how the disulfide bonds influence structural stability and flexibility of these two proteases, molecular dynamics simulations on their structures of wild-type and disulfide bond-disrupted mutant (Ver112_124C/A, Ver112_179C/A, and Ver112_124C/A_179C/A) were performed at temperatures 300 K and 400 K, respectively. Analyses of the geometrical properties along the 300 K MD trajectories indicate that PII has higher average values of Cα RMSD and solvent accessible surface area (SASA) while lower average values of number of native hydrogen bonds (NNH) and number of native contacts (NNC), suggesting a higher flexibility and less compact equilibrium structure of PII in comparison with Ver112. This may be caused by the lack of equivalent disulfide bonds in PII. The geometrical properties of Ver112 are similar on average to those of its three mutants during simulations at 300 K, while at 400 K the wild-type Ver112 presents more NHB and NNC and less SASA than its mutants suggesting that disulfide bonds contribute to the global stability of Ver112 at high temperature. Additionally, the stability of local structures within 5 Å of the two disulfide bonds C35-C124 and C179-C250 was also enhanced, as indicated by their increased RMSF and decreased NNC values upon disulfide bond breaking. Analyses of the average RMSF values of the S1 and S4 substrate-binding pockets show that upon disruption of C35-C124, RMSF of S1 pocket decreased by 21.2% while that of S4 pocket showed almost no change; upon disruption of C179-C250, the relatively large reduction in flexibility of both S1 and S4 pockets was observed; and the most pronounced reduction (30.7% and 17.2%) occurred when both disulfide bonds were broken. According to these results, we can conclude that i) the presence of disulfide bonds enhances not only the local but also the global stability of the protease, thus explaining the higher thermal stability of the alkaline protease Ver112 compared to that of the neutral protease PII; ii) the presence of disulfide bonds increases the flexibility of substrate-binding pockets located relatively far from disulfide bonds, thus explaining why alkaline proteases have higher substrate affinity (5, 6) and catalytic activity than neutral proteases.

This research was supported by grants from NSFC (No. 30860011) and Yunnan province (2007PY-22), and foundation for Key Teacher of Yunnan University.

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Lian-Ming Liang1
Ke-Qin Zhang1
Shu-Qun Liu1,2, *

1Laboratory for Conservation and Utilization of Bio-Resources & Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming 650091, P. R. China
2 Sino-Dutch Biomedial and Information Engineering School, Northeastern University, Shenyang 110003, P. R. China

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