PDBsum entry 3hq2

Hydrolase

PDB id: 3hq2

Contents

Protein chains

496 a.a.

Ligands

PO4 ×2

Metals

_CL ×7

__F

_ZN ×7

Waters ×228

References listed in PDB file

Key reference

Title

Insight into the substrate length restriction of m32 carboxypeptidases: characterization of two distinct subfamilies.

Authors

M.M.Lee, C.E.Isaza, J.D.White, R.P.Chen, G.F.Liang, H.T.He, S.I.Chan, M.K.Chan.

Ref.

Proteins, 2009, 77, 647-657. [DOI no: 10.1002/prot.22478]

PubMed id

19544567

Abstract

M32 carboxypeptidases are a distinct family of HEXXH metalloproteases whose structures exhibit a narrow substrate groove that is blocked at one end. Structural alignments with other HEXXH metalloprotease-peptide complexes suggested an orientation in which the substrate is directed towards the back of the groove. This led us to hypothesize, and subsequently confirm that the maximum substrate length for M32 carboxypeptidases is restricted. Structural and sequence analyses implicate a highly conserved Arg at the back of the groove as being critical for this length restriction. However, the Thermus thermophilus and Bacillus subtilis M32 members lack this conserved Arg. Herein, we present the biochemical and structural characterization of these two proteins. Our findings support the important role of the conserved Arg in maintaining the length restriction, and reveal a proline-rich loop as an alternate blocking strategy. Based on our results, we propose that M32 carboxypeptidases from Bacilli belong to a separate subfamily. Proteins 2009. (c) 2009 Wiley-Liss, Inc.

Figure 1.
Figure 1. Structural comparison of the subunit conformations of PfuCP (open), TthCP (open) and BsuCP (closed). (A) Ribbons diagram of PfuCP (PDB ID: 1KA2) colored in pink with the -helices of the subdomain associated with the groove closure in red. The residues forming the metal active site are shown in ball and stick. The bound Mg ion is colored in green and the remaining ions in CPK. (B) Ribbons diagram of TthCP in the same orientation colored in tan with the -helices of the subdomain associated with the conformational change in green. The residues forming the metal active site are shown in ball and stick. (C) Ribbons diagram of BsuCP subunit colored in cyan with the -helices of the subdomain associated with the conformational change in blue. The residues forming the zinc active site are shown in ball and stick. The zinc ion is colored in orange and the remaining ions in CPK. (D) Overlap of the TthCP (colored in tan and green) and BsuCP (colored in cyan and blue) structures aligned based on their common HEXXH motif. The subdomain exhibiting the largest shift is colored in green and blue, respectively. (E) Surface diagram of PfuCP showing the open conformation of the groove. The green color indicates the location of the metal active site. (F) Surface diagram of TthCP showing the open conformation of the groove. (G) Surface diagram of BsuCP showing closed groove conformation.

Figure 3.
Figure 3. Comparison of the PfuCP/BsuCP active sites with ACE2 in the open and closed conformations. Ribbons diagram of protein with side chains of key active site residues as stick drawings. The metal ion is shown as a green sphere and its bonds to its ligating residues are shown in stick. (A) PfuCP (PDB ID: 1KA4) active site in open conformation with ribbons and carbons colored in pink remaining elements in CPK. For this figure, the ring of His A411 of PfuCP was rotated by 180° to be consistent with the higher resolution TthCP structure and the orientation of the conserved residue in the BsuCP structure. (B) BsuCP active site in closed conformation with bound phosphate ion with ribbons colored in cyan. (C) ACE2 (PDB ID: 1R42) active site in open conformation in absence of bound substrate with ribbons colored in yellow.[31] (D) ACE2 (PDB ID: 1R4L) active site in closed conformation with ribbons colored in yellow and carbons of bound MLN-4760 inhibitor colored in cyan and the remaining atoms in CPK.[31] For panels (C) and (D), the ring of His A505 was rotated by 180° to allow formation of a hydrogen bond between its NE2 ring nitrogen and the Tyr A515 oxygen. The presence of this potential hydrogen bond was predicted from their contact distance ( 2.7 Å) in the 1R4L structure. This distance is long 3.3 Å in the 1R4L structure, but its His A505 was flipped as well for consistency.

The above figures are reprinted by permission from John Wiley & Sons, Inc.: Proteins (2009, 77, 647-657) copyright 2009.