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 α-lytic protease (αLP) has been solved at 0.83 Å resolution at pH 8. This ultra-high resolution structure allows accurate analysis of structural elements not possible with previous structures. Hydrogen atoms are visible, and confir...

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Veröffentlicht in:Journal of molecular biology 2004-05, Vol.338 (5), p.999-1013
Hauptverfasser: Fuhrmann, Cynthia N., Kelch, Brian A., Ota, Nobuyuki, Agard, David A.
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container_end_page 1013
container_issue 5
container_start_page 999
container_title Journal of molecular biology
container_volume 338
creator Fuhrmann, Cynthia N.
Kelch, Brian A.
Ota, Nobuyuki
Agard, David A.
description The crystal structure of the extracellular bacterial serine protease α-lytic protease (αLP) has been solved at 0.83 Å resolution at pH 8. This ultra-high 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 δ1 participates in a normal hydrogen bond with Asp102 in the catalytic triad, with a hydrogen atom visible 0.83(±0.06) Å from the His N δ1. 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 p K a of His57 is calculated to be ∼8.8 when a sulfate ion occupies the active site. This 0.83 Å 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 αLP, 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 αLP. 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.
doi_str_mv 10.1016/j.jmb.2004.03.018
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This ultra-high 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 δ1 participates in a normal hydrogen bond with Asp102 in the catalytic triad, with a hydrogen atom visible 0.83(±0.06) Å from the His N δ1. 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 p K a of His57 is calculated to be ∼8.8 when a sulfate ion occupies the active site. This 0.83 Å 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 αLP, 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 αLP. 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This ultra-high 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 δ1 participates in a normal hydrogen bond with Asp102 in the catalytic triad, with a hydrogen atom visible 0.83(±0.06) Å from the His N δ1. 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 p K a of His57 is calculated to be ∼8.8 when a sulfate ion occupies the active site. This 0.83 Å 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 αLP, 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 αLP. 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subjects atomic resolution crystallography
Binding Sites
Crystallography, X-Ray
Histidine - metabolism
Hydrogen Bonding
Models, Molecular
packing distortion
Protein Conformation
protein folding
Serine Endopeptidases - chemistry
Serine Endopeptidases - metabolism
serine protease catalysis
Sulfates - metabolism
α-lytic protease
title 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
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