Transport across cellular membranes is an essential process that is catalyzed by diverse membrane transport proteins. The turnover rates of certain transporters are inhibited by their substrates in a process termed trans-inhibition, whose structural basis is poorly understood. We present the crystal structure of a molybdate/tungstate ABC transporter (ModBC) from Methanosarcina acetivorans in a trans-inhibited state. The regulatory domains of the nucleotide-binding subunits are in close contact and provide two oxyanion binding pockets at the shared interface. By specifically binding to these pockets, molybdate or tungstate prevent adenosine triphosphatase activity and lock the transporter in an inward-facing conformation, with the catalytic motifs of the nucleotide-binding domains separated. This allosteric effect prevents the transporter from switching between the inward-facing and the outward-facing states, thus interfering with the alternating access and release mechanism.

Kumamolisin, an extracellular proteinase derived from an acido/thermophilic Bacillus, belongs to the sedolisin family of endopeptidases characterized by a subtilisin-like fold and a Ser-Glu-Asp catalytic triad. In kumamolisin, the Asp82 carboxylate hydrogen bonds to Glu32-Trp129, which might act as a proton sink stabilizing the catalytic residues. The 1.2/1.3 A crystal structures of the Glu32-->Ala and Trp129-->Ala mutants show that both mutations affect the active-site conformation, causing a 95% activity decrease. In addition, the 1.2 A crystal structure of the Ser278-->Ala mutant of pro-kumamolisin was determined. The prodomain exhibits a half-beta sandwich core docking to the catalytic domain similarly as the equivalent subtilisin prodomains in their catalytic-domain complexes. This pro-kumamolisin structure displays, for the first time, the uncleaved linker segment running across the active site and connecting the prodomain with the properly folded catalytic domain. The structure strongly points to an initial intramolecular activation cleavage in subtilases, as presumed for pro-subtilisin and pro-furin.

Corn earworm (Helicoverpa zea), also called tomato fruitworm, is a common pest of many Solanaceous plants. This insect is known to adapt to the ingestion of plant serine protease inhibitors by using digestive proteases that are insensitive to inhibition. We have now identified a B-type carboxypeptidase of H. zea (CPBHz) insensitive to potato carboxypeptidase inhibitor (PCI) in corn earworm. To elucidate the structural features leading to the adaptation of the insect enzyme, the crystal structure of the recombinant CPBHz protein was determined by x-ray diffraction. CPBHz is a member of the A͞B subfamily of metallocarboxypeptidases, which displays the characteristic metallocarboxypeptidase ␣͞␤-hydrolase fold, and does not differ essentially from the previously described Helicoverpa armigera CPA, which is very sensitive to PCI. The data provide structural insight into several functional properties of CPBHz. The high selectivity shown by CPBHz for C-terminal lysine residues is due to residue changes in the S1 substrate specificity pocket that render it unable to accommodate the side chain of an arginine. The insensitivity of CPBHz to plant inhibitors is explained by the exceptional positioning of two of the main regions that stabilize other carboxypeptidase-PCI complexes, the ␤8-␣9 loop, and ␣7 together with the ␣7-␣8 loop. The rearrangement of these two regions leads to a displacement of the active-site entrance that impairs the proper interaction with PCI. This report explains a crystal structure of an insect protease and its adaptation to defensive plant protease inhibitors.crystal structure ͉ inhibitor resistance ͉ Helicoverpa zea ͉ metallocarboxypeptidase T he larvae of plant-eating insects have a high need for free amino acids and nitrogen during their development. Normally, they meet these requirements by degrading dietary proteins. The digestive enzymes present in the larvae of the lepidopteran insects such as Helicoverpa zea are closely related to those secreted from the mammalian pancreas, comprising serine endopeptidases like trypsins and chymotrypsins and exopeptidases such as metallocarboxypeptidases and aminopeptidases (1, 2).Plants have evolved a defense that interferes with the insects' digestive system by expressing a number of different protease inhibitors (PIs), which are either constitutively present or strongly up-regulated in response to wound damage (3). Most PIs are small proteins that have been found in all plant species investigated thus far and occur in both reproductive and vegetative tissues. In herbivorous insects, they act by inhibiting protein digestive enzymes in the guts of insect larvae or adults, resulting in amino acid deficiencies that lead to serious developmental delay, mortality, or reduced fecundity.A number of polyphagous insects, among them H. zea, a common pest of many Solanaceous plants such as the potato (4), have adapted to the protease inhibitors of their various host plants. Their survival and larval development is not affected by the presence of such molecules in th...

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