X-ray analyses of aspartic proteinases. V. Structure and refinement at 2.0 A resolution of the aspartic proteinase from Mucor pusillus.
The structure of mucor pusillus pepsin (EC 3.4.23.6), the aspartic proteinase from Mucor pusillus, has been refined to a crystallographic R-factor of 16.2% at 2.0 A resolution. The positions of 2638 protein atoms, 221 solvent atoms and a sulphate ion have been determined with an estimated root-mean-square (r.m.s.) error of 0.15 to 0.20 A. In the final model, the r.m.s. deviation from ideality for bond distances is 0.022 A, and for angle distances it is 0.050 A. Comparison of the overall three-dimensional structure with other aspartic proteinases shows that mucor pusillus pepsin is as distant from the other fungal enzymes as it is from those of mammalian origin. Analysis of a rigid body shift of residues 190 to 302 shows that mucor pusillus pepsin displays one of the largest shifts relative to other aspartic proteinases (14.4 degrees relative to endothiapepsin) and that changes have occurred at the interface between the two rigid bodies to accommodate this large shift. A new sequence alignment has been obtained on the basis of the three-dimensional structure, enabling the positions of large insertions to be identified. Analysis of secondary structure shows the beta-sheet to be well conserved whereas alpha-helical elements are more variable. A new alpha-helix hN4 is formed by a six-residue insertion between positions 131 and 132. Most insertions occur in loop regions: -5 to 1 (five residues relative to porcine pepsin): 115 to 116 (six residues); 186 to 187 (four residues); 263 to 264 (seven residues); 278 to 279 (four residues); and 326 to 332 (six residues). The active site residues are highly conserved in mucor pusillus pepsin; r.m.s. difference with rhizopuspepsin is 0.37 A for 25 C alpha atom pairs. However, residue 303, which is generally conserved as an aspartate, is changed to an asparagine in mucor pusillus pepsin, possibly influencing pH optimum. Substantial changes have occurred in the substrate binding cleft in the region of S1 and S3 due to the insertion between 115 and 116 and the rearrangement of loop 9-13. Residue Asn219 necessitates a shift in position of substrate main-chain atoms to maintain hydrogen bonding pattern. Invariant residues Asp11 and Tyr14 have undergone a major change in conformation apparently due to localized changes in molecular structure. Both these residues have been implicated in zymogen stability and activation.
Newman
M
M
Department of Crystallography, Birkbeck College, University of London, U.K.
Watson
F
F
Roychowdhury
P
P
Jones
H
H
Badasso
M
M
Cleasby
A
A
Wood
S P
SP
Tickle
I J
IJ
Blundell
T L
TL
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000648:ultrastructure*; D001665:Binding Sites; D003461:Crystallography; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000648:ultrastructure*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology*; D010434:Pepsin A / Q000648:ultrastructure*; D011498:Protein Precursors / Q000648:ultrastructure; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D012997:Solvents; D014961:X-Ray Diffraction
D005656:Fungal Proteins; D011498:Protein Precursors; D012997:Solvents; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A