The crystal structures of two homologous inhibitors (PMP-C and PMP-D2v) from the insect Locusta migratoria have been determined in complex with bovine ␣-chymotrypsin at 2.1-and 3.0-Å resolution, respectively. PMP-C is a potent bovine ␣-chymotrypsin inhibitor whereas native PMP-D2 is a weak inhibitor of bovine trypsin. One unique mutation at the P1 position converts PMP-D2 into a potent bovine ␣-chymotrypsin inhibitor. The two peptides have a similar overall conformation, which consists of a triple-stranded antiparallel ␤-sheet connected by three disulfide bridges, thus defining a novel family of serine protease inhibitors. They have in common the protease interaction site, which is composed of the classical protease binding loop (position P5 to P4, corresponding to residues 26 -34) and of an internal segment (residues 15-18), held together by two disulfide bridges. Structural divergences between the two inhibitors result in an additional interaction site between PMP-D2v (position P10 to P6, residues 21-25) and the residues 172-175 of ␣-chymotrypsin. This unusual interaction may be responsible for species selectivity. A careful comparison of data on bound and free inhibitors (from this study and previous NMR studies, respectively) suggests that complexation to the protease stabilizes the flexible binding loop (from P5 to P4).Small canonical serine protease inhibitors are widely distributed among living organisms. They have been classified by Bode and Huber (1) into 16 structural families. One of the novel families that has emerged since then is the grasshopper family (2). The first members were characterized from the brain (pars intercerebralis) and the hemolymph of the insect Locusta migratoria (3-5). Furthermore, similar peptides (named SGPI) were isolated from Schistocerca gregaria (6); they share 40 -80% homology (including the six conserved cysteines) with the Locusta peptides. More recently, the same sequence motif was also identified in Pacifastin, a 155-kDa protein from the crayfish Pacifastacus leniusculus and composed of two domains with different activities. One of these domains contains nine repeats similar to the locust peptides and has a protease inhibitory activity (7).We have carried out extensive investigations on two locust peptides, PMP-C and PMP-D2 (pars intercerebralis major peptide). They consist of 36 and 35 residues, respectively, have three disulfide bridges, and are 40% identical in sequence. The three-dimensional structures of PMP-D2 and PMP-C were determined by 1 H NMR (8, 9). These peptides display a new fold with an unusual disulfide bond pattern. PMP-C is a very potent bovine ␣-chymotrypsin inhibitor (K i 0.13 nM) and a weak human leukocyte elastase inhibitor (K i 180 nM) and is devoid of activity toward porcine trypsin. The nature of its P1 residue (nomenclature according to Schechter and Berger (10)), Leu 30 , is in accordance with the literature and its inhibitory properties. Indeed, chymotrypsin inhibitors have bulky and aromatic residues such as Phe, Leu, or Met as their P1 res...

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The three-dimensional structures of two cytochromes c' have been determined in order to analyse the common features of proteins of this family and their relationship with other four-helix bundle structures. The structure of cytochrome c' from Alcaligenes sp was determined by molecular replacement supplemented with the iron anomalous scattering and the use of a single isomorphous heavy-atom derivative, and was refined using synchrotron data to 1.8 A resolution. The final model, comprising 956 protein atoms (one monomer) and 89 water molecules, has a final R value of 0.188 for all data in the range 20.0-1.8 A resolution (14 673 reflections). The structure of the cytochrome c' from Alcaligenes denitrificans is isomorphous and essentially identical (r.m.s. deviation for all atoms 0.36 A). Although its amino-acid sequence has not been determined chemically, only four differences from that of Alcaligenes sp cytochrome c' were identified by the X-ray analysis. The final model for Alcaligenes denitrificans cytochrome c', comprising 953 protein atoms and 75 water molecules, gave a final R factor of 0.167 for all data in the range 20.0-2.15 A (8220 reflections). The cytochrome c' monomer forms a classic four-helix bundle, determined by the packing of hydrophobic side chains around the enclosed haem group. There are very few cross-linking hydrogen bonds between the helices, the principal side-chain hydrogen bonding involving one of the haem propionates and a conserved Arg residue. The cytochrome c' dimer is created by a crystallographic twofold axis. Monomer-monomer contacts primarily involve the two A helices, with size complementarity of side chains in a central solvent-excluded portion of the interface and hydrogen bonding at the periphery. Both species have a pyroglutamic acid N-terminal residue. The haem iron is five-coordinate, 0.32 A out of the haem plane towards the fifth ligand, His120. The unusual magnetic properties of the Fe atom may be linked to a conserved basic residue, Arg124, adjacent to His120.

The structure of cytochrome c' from two bacterial species, Alcaligenes sp and Alcaligenes denitrificans, have been determined from X-ray diffraction data to 3.0 A resolution using the anomalous scattering of the single Fe atom in each to identify and ref'me a weak molecular-replacement solution. Molecular-replacement studies, with the program AMORE, used two isomorphous data sets (from the two species), two independent search models (the cytochromes c' from Rhodospirillum molischianum and Rhodospirillum rubrum), both with and without side chains, and two different resolution ranges (10.0-4.0 and 15.0--3.5A) to generate a large number of potential solutions. No single solution stood out and none appeared consistently. The Fe-atom position in each structure was then determined from its anomalous-scattering contribution and all molecularreplacement solutions were discarded which did not (i) place the Fe atom correctly and (ii) orient the molecule such that a crystallographic twofold axis generated a dimer like those of the two search models. Finally, electron-density maps phased solely by the Fe-atom anomalous scattering were calculated. As these were combined and subjected to solvent flattening and histogram matching (with the program SQUASH), correlation with the remaining molecular-replacement solutions identified one as correct and enabled it to be improved and subjected to preliminary refinement. The correctness of the solution is confirmed by parallel isomorphous-replacement studies.

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