F. X. Avilés

Three-dimensional structure of porcine procarboxypeptidase B: a structural basis of its inactivity.

Procarboxypeptidase B is converted to enzymatically active carboxypeptidase B by limited proteolysis catalysed by trypsin, removing the long N‐terminal activation segment of 95 amino acids. The three‐dimensional crystal structure of procarboxypeptidase B from porcine pancreas has been determined at 2.3 A resolution and refined to a crystallographic R‐factor of 0.169. The functional determinants of its enzymatic inactivity and of its activation by limited proteolysis have thus been unveiled. The activation segment folds in a globular region with an open sandwich antiparallel‐alpha antiparallel‐beta topology and in a C terminal alpha‐helix which connects it to the enzyme moiety. The globular region (A7‐A82) shields the preformed active site, and establishes specific interactions with residues important for substrate recognition. AspA41 forms a salt bridge with Arg145, which in active carboxypeptidase binds the C‐terminal carboxyl group of substrate molecules. The connecting region occupies the putative extended substrate binding site. The scissile peptide bond cleaved by trypsin during activation is very exposed. Its cleavage leads to the release of the activation segment and to exposure of the substrate binding site. An open‐sandwich folding has been observed in a number of other proteins and protein domains. One of them is the C‐terminal fragment of L7/L12, a ribosomal protein from Escherichia coli that displays a topology similar to the activation domain of procarboxypeptidase.

Crystal Structure of Avian Carboxypeptidase D Domain II : A Prototype for the Regulatory Metallocarboxypeptidase Subfamily

The crystal structure of domain II of duck carboxypeptidase D, a prohormone/propeptide processing enzyme integrated in a three repeat tandem in the natural system, has been solved, constituting a prototype for members of the regulatory metallocarboxypeptidase subfamily. It displays a 300 residue N‐terminal α/β‐hydrolase subdomain with overall topological similarity to and general coincidence of the key catalytic residues with the archetypal pancreatic carboxypeptidase A. However, numerous significant insertions/deletions in segments forming the funnel‐like access to the active site explain differences in specificity towards larger protein substrates or inhibitors. This α/β‐hydrolase subdomain is followed by a C‐terminal 80 residue β‐sandwich subdomain, unique for these regulatory metalloenzymes and topologically related to transthyretin and sugar‐binding proteins. The structure described here establishes the fundamentals for a better understanding of the mechanism ruling events such as prohormone processing and will enable modelling of regulatory carboxypeptidases as well as a more rational design of inhibitors of carboxypeptidase D.

THE THREE-DIMENSIONAL STRUCTURE OF THE NATIVE TERNARY COMPLEX OF BOVINE PANCREATIC PROCARBOXYPEPTIDASE A WITH PROPROTEINASE E AND CHYMOTRYPSINOGEN C

The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far.

Mammalian metallopeptidase inhibition at the defense barrier of Ascaris parasite

Roundworms of the genus Ascaris are common parasites of the human gastrointestinal tract. A battery of selective inhibitors protects them from host enzymes and the immune system. Here, a metallocarboxypeptidase (MCP) inhibitor, ACI, was identified in protein extracts from Ascaris by intensity-fading MALDI-TOF mass spectrometry. The 67-residue amino acid sequence of ACI showed no significant homology with any known protein. Heterologous overexpression and purification of ACI rendered a functional molecule with nanomolar equilibrium dissociation constants against MCPs, which denoted a preference for digestive and mast cell A/B-type MCPs. Western blotting and immunohistochemistry located ACI in the body wall, intestine, female reproductive tract, and fertilized eggs of Ascaris, in accordance with its target specificity. The crystal structure of the complex of ACI with human carboxypeptidase A1, one of its potential targets in vivo, revealed a protein with a fold consisting of two tandem homologous domains, each containing a β-ribbon and two disulfide bonds. These domains are connected by an α-helical segment and a fifth disulfide bond. Binding and inhibition are exerted by the C-terminal tail, which enters the funnel-like active-site cavity of the enzyme and approaches the catalytic zinc ion. The findings reported provide a basis for the biological function of ACI, which may be essential for parasitic survival during infection.

Insights into the molecular inactivation mechanism of human activated thrombin-activatable fibrinolysis inhibitor

See also Gils A. Hot spots in TAFIa. This issue, pp 1054–5.

Structure and dynamics of the potato carboxypeptidase inhibitor by 1H and 15N NMR.

The solution structure and backbone dynamics of the recombinant potato carboxypeptidase inhibitor (PCI) have been characterized by NMR spectroscopy. The structure, determined on the basis of 497 NOE‐derived distance constraints, is much better defined than the one reported in a previous NMR study, with an average pairwise backbone root‐mean‐square deviation of 0.5 Å for the well‐defined region of the protein, residues 7–37. Many of the side‐chains show now well‐defined conformations, both in the hydrophobic core and on the surface of the protein. Overall, the solution structure of free PCI is similar to the one that it shows in the crystal of the complex with carboxypeptidase A. However, some local differences are observed in regions 15–21 and 27–29. In solution, the six N‐terminal and the two C‐terminal residues are rather flexible, as shown by 15N backbone relaxation measurements. The flexibility of the latter segment may have implications in the binding of the inhibitor by the enzyme. All the remaining residues in the protein are essentially rigid (S2 > 0.8) with the exception of two of them at the end of a short 3/10 helix. Despite the small size of the protein, a number of amide protons are protected from exchange with solvent deuterons. The slowest exchanging protons are those in a small two‐strand β‐sheet. The unfolding free energies, as calculated from the exchange rates of these protons, are around 5 kcal/mol. Other protected amide protons are located in the segment 7–12, adjacent to the β‐sheet. Although these residues are not in an extended conformation in PCI, the equivalent residues in structurally homologous proteins form a third strand of the central β‐sheet. The amide protons in the 3/10 helix are only marginally protected, indicating that they exchange by a local unfolding mechanism, which is consistent with the increase in flexibility shown by some of its residues. Backbone alignment‐based programs for folding recognition, as opposite to disulfide‐bond alignments, reveal new proteins of unrelated sequence and function with a similar structure. Proteins 2003;50:410–422.

Comparison of the NMR solution structure with the X-ray crystal structure of the activation domain from procarboxypeptidase B

SummaryThe NMR solution structure of the activation domain isolated from porcine procarboxypeptidase B is compared with the X-ray crystal structure of the corresponding segment in the intact proenzyme. For the region of the polypeptide chain that has a well-defined three-dimensional structure in solution, i.e., the backbone atoms of residues 11–76 and 25 amino acid side chains in this segment that form a hydrophobic core in the activation domain, the root-mean-square distance between the two structures is 1.1 Å. There are no significant differences in average atom positions between the two structures, but only the NMR structure shows increased structural disorder in three outlying loops located along the same edge of the activation domain. These regions of increased structural disorder in the free domain coincide only partially with the interface to the enzyme domain in the proenzyme.

Overproduction of a recombinant carboxypeptidase inhibitor by optimization of fermentation conditions

In order to optimize the production of recombinant potato carboxypeptidase inhibitor (rePCI), a protein with 39 amino acid residues and three disulphide bridges, by Escherichia coli MC1061[pIMAM3], the effects of various parameters were investigated. Production of rePCI in M9CAS medium was optimal at 37°C and using low concentrations of glycerol as a carbon source. Increasing concentrations of glycerol caused a decrease in the production of rePCI, which was almost totally inhibited above 1% glycerol. Relatively high concentrations of oligoelements in the culture medium also inhibited the production of rePCI. We obtained a 100-fold increase in the production of rePCI, from 2 to 200 mg/l, when growing bacteria in a fed-batch aerobic culture using a 2-1 fermentor. RePCI was found exclusively in the supernatant, although the genetic construction was designed for it to be released into the periplasmic space. Large quantities of rePCI could be easily purified from the supernatants of these cultures. Our conditions of fed-batch, aerobic fermentation could be used for overproduction to high levels of other recombinant proteins.

Structure and atomic fluctuation patterns of potato carboxypeptidase a inhibitor protein

Molecular dynamics (MD) simulation methods were applied to the study of the structural and dynamic fluctuation properties of the potato carboxypeptidase A inhibitor protein (PCI) immersed in a bath of 1259 water molecules. A trajectory of 200 ps was generated at constant temperature and pressure. The crystallographic structure of PCI, as found in its complex with bovine carboxy-peptidase A (CPA), was used to seed the MD simulation. Analyses show that the structure of the PCI core is fairly rigid and stable in itself, and that little deformation is caused by the protein-protein interactions found in the PCI-CPA complex. The N-terminal tail fluctuates to approach the core structure and appears as a relatively disordered region. In contrast, the conformations of the C-terminal tail, which is involved in the inhibitory mechanism, fluctuates in the neighborhood of the X-ray structure in orientations which facilitate CPA binding. Comparison with the structural entries for PCI in water obtained from both 2D-NMR experiments and X-ray data shows that important features of the MD structural results fluctuates between the initial crystal values and those obtained from the NMR solution structure. This fluctuation is not uniform; minor regions move away from the X-ray conformation while they do not approach the NMR conformation. The results reported suggest that the trajectory is long enough to show a behavior that is consistent with the conformational space available to the protein in solution.

Determination of hemihedral twinning and initial structural analysis of crystals of the procarboxypeptidase A ternary complex

The initial structural analysis of the ternary complex of procarboxypeptidase A from hemihedrally twinned crystals diffracting up to 2.8 A is described. Detection of twinning by different techniques is presented, including biochemical and intensity statistics approaches. The structure was initially solved using Patterson-search techniques, and the three positioned search models were used to effectively deconvolute the twinned data.