Enzyme specificity under dynamic control II: Principal component analysis of alpha-lytic protease using global and local solvent boundary conditions

Ota, N Agard, DA

N. Ota et Da. Agard, Enzyme specificity under dynamic control II: Principal component analysis of alpha-lytic protease using global and local solvent boundary conditions, PROTEIN SCI, 10(7), 2001, pp. 1403-1414

50

INGLESE

Article

Biochemistry & Biophysics

PROTEIN SCIENCE

0961-8368 → ACNP

10

7

2001

1403 - 1414

ISI

0961-8368(200107)10:7<1403:ESUDCI>2.0.ZU;2-5

The contributions of conformational dynamics to substrate specificity have been examined by the application of principal component analysis to molecul ar dynamics trajectories of alpha -lytic protease. The wild-type alpha -lyt ic protease is highly specific for substrates with small hydrophobic side c hains at the specificity pocket, while the Metl90 --> Ala binding pocket mu tant has a much broader specificity, actively hydrolyzing substrates rangin g from Ala to Phe. Based on a combination of multiconformation analysis of cryo-X-ray crystallographic data, solution nuclear magnetic resonance (NMR) , and normal mode calculations, we had hypothesized that the large alterati on in specificity of the mutant enzyme is mainly attributable to changes in the dynamic movement of the two walls of the specificity pocket. To test t his hypothesis, we performed a principal component analysis using I-nanosec ond molecular dynamics simulations using either a global or local solvent b oundary condition. The results of this analysis strongly support our hypoth esis and verify the results previously obtained by in vacuo normal mode ana lysis. We found that the walls of the wild-type substrate binding pocket mo ve in tandem with one another, causing the pocket size to remain fixed so t hat only small substrates are recognized. In contrast, the M190A mutant sho ws uncoupled movement of the binding pocket walls, allowing the pocket to s ample both smaller and larger sizes, which appears to be the cause of the o bserved broad specificity. The results suggest that the protein dynamics of ol-lytic protease may play a significant role in defining the patterns of substrate specificity. As shown here, concerted local movements within prot eins can be efficiently analyzed through a combination of principal compone nt analysis and molecular dynamics trajectories using a local solvent bound ary condition to reduce computational time and matrix size.