Crystal structure of papain-succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide complex at 1.7-A resolution: noncovalent binding mode of a common sequence of endogenous thiol protease inhibitors.

Succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide corresponding to a common sequence of endogenous thiol protease inhibitors is a noncompetitive reversible inhibitor of papain. In order to elucidate the binding mode of the inhibitor at the atomic level, its complex with papain was crystallized at ca. pH 7.0 using the hanging drop method, and the crystal structure was analyzed at 1.7-A resolution. The crystal has space group P2(1)2(1)2(1), with a = 43.09, b = 102.32, c = 49.69 A, and Z = 4. A total of 47,215 observed reflections were collected on the imaging plates using the same single crystal, and 19,833 unique reflections with Fo > sigma (Fo) were used for structure determination and refinement. The papain structure was determined by use of the atomic coordinates of papain previously reported, and then refined by the X-PLOR program. The inhibitor molecule was located on a difference Fourier map and fitted into the electron density with the aid of computer graphics. The complex structure was finally refined to R = 19.6% including 118 solvent molecules. The X-ray analysis of the complex crystal shows that the inhibitor is located at the R-domain side, not in the center of the binding site created by the R- and L-domains of papain. Such a binding mode of the inhibitor explains well the biological behavior that the inhibitor exhibits against papain. Comparison with the structure of papain-stefin B complex indicates that the structure of the Gln-Val-Val-Ala-Gly sequence itself is not necessarily the essential requisite for inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)