The 0.83 Å resolution crystal structure of α-lytic protease reveals the detailed structure of the active site and identifies a source of conformational strain

The crystal structure of the extracellular bacterial. serine protease alpha-lytic of protease (alphaLP) has been solved at 0.83 Angstrom resolution at pH 8. This ultrahigh resolution structure allows accurate analysis of structural elements not possible with previous structures. Hydrogen atoms are visible, and confirm active-site hydrogen-bonding interactions expected for the apo enzyme. In particular, His57 N-delta1 participates in a normal hydrogen bond with Asp102 in the catalytic triad, with a hydrogen atom visible 0.83( +/- 0.06) Angstrom from the His N-delta1. The catalytic Ser195 occupies two conformations, one corresponding to a population of His57 that is doubly protonated, the other to the singly protonated His57. Based on the occupancy of these conformations, the pK(a) of His57 is calculated to be similar to8.8 when a sulfate ion occupies the active site. This 0.83 A structure has allowed critical analysis of geometric distortions within the structure. Interestingly, Phe228 is significantly distorted from planarity. The distortion of Phe228, buried in the core of the C-terminal domain, occurs at an estimated energetic cost of 4.1 kcal/mol. The conformational space for Phe228 is severely limited by the presence of Trp199, which prevents Phe228 from adopting the rotamer observed in many other chymotrypsin family members. In alphaLP, the only allowed rotamer leads to the deformation of Phe228 due to steric interactions with Thr181. We hypothesize that tight packing of co-evolved residues in this region, and the subsequent deformation of Phe228, contributes to the high cooperativity and large energetic barriers for folding and unfolding of alphaLP. The kinetic stability imparted by the large, cooperative unfolding barrier plays a critical role in extending the lifetime of the protease in its harsh environment. (C) 2004 Elsevier Ltd. All rights reserved.