A peptide inhibitor, having the sequence D-His-Pro-Phe-His-PheI[CH2-NH]Phe-Val-Tyr, with a reduced bond between the two adjacent phenylalanines, has been diffused into crystals of the aspartic proteinase from Rhizopus chinensis (rhizopuspepsin, EC 3.4.23.6). X-ray diffraction data to 1.8-A resolution have been collected on the complex, which has been subjected to restrained least-squares refinement to an R-factor (R = Z;|1FO1 -FII[IFOI, where IFOI and JFJ are the observed and calculated structure factor amplitudes, respectively) of 14.7%. The inhibitor lies within the major groove of the enzyme and is clearly defined with the exception of the amino-terminal D-histidine and the carboxyl-terminal tyrosine.The reduced peptide bond is located in the active site with close contacts to the two catalytic aspartyl groups. The active-site water molecule that is held between the two carboxyl groups is displaced by the inhibitor, as are a number of other water molecules seen in the binding groove of the native enzyme. A mechanism of action for this class of enzymes is proposed from these results.The aspartic proteinases, which include the mammalian enzymes pepsin, gastricsin, chymosin, cathepsin D, and renin, as well as a number of microbial and plant enzymes, form a class of digestive enzymes having several common properties. They are optimally active at acidic pH, have two aspartic acids at the active site, and are all inactivated by certain inhibitors, such as pepstatin. In addition, they all display subsite specificities extending for several residues on either side of the scissile bond. Detailed x-ray analyses for four of these enzymes-namely, pepsin and three fungal enzymes, penicillopepsin, endothiapepsin and rhizopuspepsin [refs. 1, 2 (pp. 137-150), 3-8] have shown very similar three-dimensional structures; this similarity is particularly evident in the enzyme active sites. The mechanism of action of these proteinases has been extensively discussed (for review, see ref.2). Earlier studies provided no compelling evidence for the formation of a covalent intermediate during catalysis (9,10). Consequently, recent mechanistic hypotheses based on the three-dimensional structures of these enzymes have invoked nucleophilic attack by a water molecule on the carbonyl carbon, with possible intervention of the carboxyl groups of the enzyme in transferring a proton to the substrate amino group [refs. 2 (pp. 189-195), 11, 12]. However, the identity of this water molecule remains unresolved.We describe an 1.8-A analysis of the complex of rhizopuspepsin with a reduced peptide inhibitor of the sequence D-His-Pro-Phe-His-PheT[CH2-NH]Phe-Val-Tyr [nomenclature of Spatola (13)] and relate these results to a mechanism of action for aspartic proteinases. MATERIALS AND METHODSCrystals of the native enzyme were grown as described (8). Intensity data were collected using the Mark II multiwire area detector system at the University of California at San Diego [Resource for Protein Crystallography, National Institutes of Health Grant...

The structure of the complex of the tetrameric peanut lectin with lactose has been refined to an R-value of 16.4% using 2.25 Å resolution X-ray Indian Institute of Science diffraction data. The subunit conformation in the structure is similar to that Bangalore-560 012, India in other legume lectins except in the loops. It has been shown that in the tertiary structure of legume lectins, the short five-stranded sheet plays a major role in connecting the larger flat six-stranded and curved seven-stranded sheets. Furthermore, the loops that connect the strands at the two ends of the seven-stranded sheet curve toward and interact with each other to produce a second hydrophobic core in addition to the one between the two large sheets. The protein-lactose interactions involve the invariant features observed in other legume lectins in addition to those characteristic of peanut lectin. The ''open'' quaternary association in peanut lectin is stabilised by hydrophobic, hydrogen-bonded and water-mediated interactions. Contrary to the earlier belief, the structure of peanut lectin demonstrates that the variability in quaternary association in legume lectins, despite all of them having nearly the same tertiary structure, is not necessarily caused by covalently bound carbohydrate. An attempt has been made to provide a structural rationale for this variability, on the basis of buried surface areas during dimerisation. A total of 45 water molecules remain invariant when the hydration shells of the four subunits are compared. A majority of them appear to be involved in stabilising loops.

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Rotavirus NSP4 is a multifunctional endoplasmic reticulum (ER)-resident nonstructural protein with the N terminus anchored in the ER and about 131 amino acids (aa) of the C-terminal tail (CT) oriented in the cytoplasm. Previous studies showed a peptide spanning aa 114 to 135 to induce diarrhea in newborn mouse pups with the 50% diarrheal dose approximately 100-fold higher than that for the full-length protein, suggesting a role for other regions in the protein in potentiating its diarrhea-inducing ability. In this report, employing a large number of methods and deletion and amino acid substitution mutants, we provide evidence for the cooperation between the extreme C terminus and a putative amphipathic ␣-helix located between aa 73 and 85 (AAH [73][74][75][76][77][78][79][80][81][82][83][84][85] ) at the N terminus of ⌬N72, a mutant that lacked the N-terminal 72 aa of nonstructural protein 4 (NSP4) from Hg18 and SA11. Cooperation between the two termini appears to generate a unique conformational state, specifically recognized by thioflavin T, that promoted efficient multimerization of the oligomer into high-molecular-mass soluble complexes and dramatically enhanced resistance against trypsin digestion, enterotoxin activity of the diarrhea-inducing region (DIR), and double-layered particle-binding activity of the protein. Mutations in either the C terminus, AAH 73-85 , or the DIR resulted in severely compromised biological functions, suggesting that the properties of NSP4 are subject to modulation by a single and/or overlapping highly sensitive conformational domain that appears to encompass the entire CT. Our results provide for the first time, in the absence of a three-dimensional structure, a unique conformation-dependent mechanism for understanding the NSP4-mediated pleiotropic properties including virus virulence and morphogenesis.Rotavirus is the most common cause of life-threatening, severe dehydrating diarrhea in children and animals (50). Rotavirus infection can be either symptomatic or asymptomatic. But the genetic/molecular basis for rotavirus virulence is not yet clearly understood. The recent identification of the nonstructural protein 4 (NSP4) as the first viral enterotoxin has attracted considerable attention toward understanding its structure and function. But analysis of NSP4 sequences from more than 175 strains failed to reveal any sequence motifs or amino acids that segregated with the virulence phenotype of the virus. Furthermore, a peptide spanning amino acids (aa) 114 to 135 was reported to induce diarrhea at an approximately 100-fold molar excess compared to the full-length protein (6). This suggested that other regions in the protein might influence its diarrhea-inducing potential. Also, the extreme C terminus, including the terminal methionine, was shown to be important for double-layered particle (DLP)-binding activity. NSP4 is 175 aa in length, with the N-terminal region anchored in the endoplasmic reticulum (ER) and approximately 131 aa of the C terminus oriented in the cytoplasm. The...

The x-ray crystal structure of the tetrameric T-antigen-binding lectin from peanut, Mr 110,000, has been determined by using the multiple isomorphous replacement method and refined to an R value of 0.218 for 22,155 reflections within the 10-to 2.95-resolution range. Each subunit has essentially the same characteristic tertiary fold that is found in other legume lectins. The structure, however, exhibits an unusual quaternary arrangement of subunits. Unlike other well-characterized tetrameric proteins with identical subunits, peanut lectin has neither 222 (D2) nor fourfold (C4) symmetry. A noncrystallographic twofold axis relates two halves of the molecule. The two monomers in each half are related by a local twofold axis. The mutual disposition of the axes is such that they do not lead to a closed point group. Furthermore, the structure of peanut lectin demonstrates that differences in subunit arrangement in legume lectins could be due to factors intrinsic to the protein molecule and, contrary to earlier suggestions, are not necessarily caused by interactions involving covalently linked sugar. The structure provides a useful framework for exploring the structural basis and the functional implications of the variability in the subunit arrangement in legume lectins despite all of them having nearly the same subunit structure, and also for investigating the general problem of "open" quaternary assembly in oligomeric proteins.Lectins are multivalent proteins of nonimmune origin that bind cell surface carbohydrates with high specificity (1, 2). Although originally isolated from plant sources and characterized by their ability to agglutinate erythrocytes, lectins are now known to be ubiquitous in nature, with binding specificities for a wide variety of cells. They have received considerable attention in recent years on account of their use in studies on biological receptors and cell surface phenomena.The most extensively studied lectins are those obtained from the seeds of leguminous plants. These lectins are either dimeric or tetrameric. The first lectin to be x-ray analyzed, in the 1970s, was the tetrameric concanavalin A (Con A) from thejack bean (3, 4). The three-dimensional structures ofthree more lectins, those of pea lectin, favin, and isolectin I from the seeds ofLathyrus ochrus, became available subsequently (5-7). In the meantime, it was shown by several workers that legume lectins are related to one another by sequence homology (8). As is to be expected from the homology, the subunits in Con A, pea lectin, favin, and the L. ochrus lectin have nearly the same tertiary structure, although Con A has a single-chain subunit while the other three have two polypeptide chains in each subunit. They contain two metal ions each (calcium and manganese), which are situated in the same locations in the three-dimensional structures of the four lectins. The locations of the carbohydrate-binding region in them are also broadly similar. Furthermore, the lectin subunits dimerize in a similar fashion. The dimers further...