Serine proteases 3 sheet

CI2 (Figure 19.2) is a 64-residue polypeptide inhibitor of serine proteases.23 It has a binding loop (Met-40, which binds in the primary site of chymotrypsin or subtilisin), a single a helix running from residues 12 to 24, and a mixed parallel and antiparallel /3 sheet. The strands and the amphipathic helix interact to form... 

Orthogonal fl-Sheet Proteins. An alternative way of packing two [3 sheets together is with the strands in the two sheets almost perpendicular. Each domain of the serine proteases (e.g., trypsin, Figure 12.Id) shows this arrangement. 

The cereal dual function a-amylase/trypsin inhibitor proteins are cysteine-rich, disulphide-rich, double-headed, 13-16 kDa, dual function inhibitor proteins that inhibit both of the digestion enzymes a-amylase and trypsin [290-325] (Table 11). Thus the Zea (com) member of this family, com Hageman factor inhibitor (CHFI), is a double-headed 14 kDa protein that inhibits a-amylase and the serine proteases trypsin and blood clotting Factor Xlla [323-324] (Table 11). The structures of the bifunctional a-amylase/trypsin inhibitor proteins from Eleusine (ragi) (RBI) [292-295] and Zea (com) (CHFI) [325] have been determined. These proteins are structurally similar to the lipid transfer proteins, being composed of a bundle of 4 a-helices together with a short [3-sheet element connected by loops, the a-amylase- and protease-inhibitory domains being separately located [325]. 

The formation of a /3-sheet has been frequently observed in protein-peptide interactions, such as substrate recognition by certain serine proteases (Tong et at, 1998) and peptide recognition by the PTB and PDZ domains (Kuriyan and Cowbum, 1997). Detailed analysis further revealed that the central portion of the receptor peptide (P 2> Po>... 

Fig. 7.1 The tetramer of eco bound to a serine protease. Visualized as a cartoon of the canonical protease and eco interaction (a), and (b), as two views of the three dimensional solution of D102N trypsin in complex with eco [3]. Each eco molecule has three protein-protein interaction surfaces. The C-terminus forms an anti-parallel p ribbon to complete the ecotin dimer interface. The 80 s and 50 s loops form the primary binding site by interacting with the protease at the active site cleft in a sub-strate-like y -sheet conformation. The 60 s and lOO s loops of eco form the secondary binding site by interacting with the C-termi-nal a-helix of the protease. Note that each eco molecule contacts both of the protease molecules. Two eco molecules (black and medium grey) form a pair of interactions each with two protease molecules (light grey). The catalytic triad residues Ser-195, Asp-102 and His-57 are in black ball and stick representation. This figure was made with Molscript [37] and Raster 3D [38].

A protein S, the cofactor of activated protein C, has four EGF-like modules in tandem. The stracture of N-terminal EGF modules is very similar to that of non-calcium-binding modules. Calcium binding only results in a limited local conformational change. The N-terminal loop is better defined and moves toward the major /3-sheet. The N-terminal non-catalytic Gla and EGF-like domains provide coagulation serine proteases with different calcium affinities for certain biological membranes, and also mediate protein-protein interactions. ... 

The most thoroughly studied mechanism of protein protease inhibitors is that of the standard mechanism (or Canonical or Laskowski mechanism) inhibitors of serine proteases (1) (Fig. 2). Standard mechanism inhibitors include the Kazal, Kunitz, and Bowman-Birk family of inhibitors and bind in a lock-and-key fashion. Ah standard mechanism inhibitors insert a reactive loop into the active site of the protease, which is complementary to the substrate specificity of the target protease and binds in an extended fi-sheet with the enzyme in a substrate-like manner. WhUe bound to the protease, the scissile bond of standard mechaiusm inhibitors is hydrolyzed very slowly, but products are not released and the amide bond is re-ligated. The standard mechanism is an efficient way to inhibit serine proteases, and it is thus used by many structurally... 

Serpins consist of a conserved core of three P-sheets and eight or nine a-helices that act collectively in the inhibitory mechanism. As with the Kazal- and Kunitz-type inhibitors, the mechanism involves a surface exposed loop that is termed the reactive center loop (RCL). The RCL presents a short stretch of polypeptide sequence bearing the Pl-Pl scissile bond. Like other serine protease inhibitor families, the PI residue dominates the thermodynamics that govern the interaction between protease and inhibitor. Exposure of the PI residue to solvent is typically brokered by 15 amino acids N-terminal to the PI residue and 5 residues on the C-terminal prime side of the scissile bond. Evidence for dramatic conformational change in the inhibitory mechanism was first provided by the crystal structure of the cleaved form of ai-antitrypsin (37). In this structure and unlike the native form, the reactive center loop was not solvent exposed but occurred as an additional P-strand within the core of the structure. 

If thrombin and factor Xa, the major activated blood coagulation factors (Fig. 11.6), escape into healthy blood vessels, blood clots will develop and occlude capillaries throughout the body. Direct inhibition of these activated enzymes in the blood flow utilizes serine protease inhibitors, of which there are two common types a Kunitz inhibitor and a serpin. The former possess a Kunitz domain, a convex antiparallel (1-sheet that exactly fits into the concave active site of a serine protease, directly blocking it (lock and key mechanism). By contrast, serpins undergo complex interactions with other proteins to cause conformational changes that bait and block the catalytic action (Fig. 11.12 shows the bait). Table 11.3 fists the major coagulation inhibitors and cofactors, their targets and mechanisms of action. 

The DhiA enzyme functions as a monomer ( 35 kDa) and is composed of two domains a main domain and a cap domain (Figure 2(a)). The main domain consists of a mostly parallel eight-stranded /3-sheet connected by ct-helices on both sides of the sheet. The cap domain is composed of five ct-helices with intervening loops. The active site is an occluded hydrophobic cavity located at the interface of the two domains. The overall fold of the main domain is the hallmark of the o //3-hydrolase fold superfamily of enzymes, to which lipases, esterases, carboxypeptidases, and acetylcholinesterases also belong. These superfamily members catalyze the hydrolysis of ester and amide bonds via a two-step nucleophilic substitution mechanism similar to that of serine proteases. 

However, Sielecki and co-workers (1979) have not found deviations from planarity, or any preference for the sign of Agi, in the 179 trans peptide bonds considered in the refined three-dimensional structure of Streptomyces griseus serine protease A at 1.8 A resolution. They concluded that deformation of the peptide bond does not significantly determine chirality of P sheets. 

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