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Maltodextrin glucosidase (MalZ) is a key enzyme in the maltose utilization pathway in Escherichia coli that liberates glucose from the reducing end of the short malto-oligosaccharides. Unlike other enzymes in the GH13_21 subfamily, the hydrolytic activity of MalZ is limited to maltodextrin rather than long starch substrates, forming various transglycosylation products in α-1,3, α-1,4 or α-1,6 linkages. The mechanism for the substrate binding and hydrolysis of this enzyme is not well understood yet. Here, we present the dimeric crystal structure of MalZ, with the N-domain generating a unique substrate binding groove. The N-domain bears CBM34 architecture and forms a part of the active site in the catalytic domain of the adjacent molecule. The groove found between the N-domain and catalytic domain from the adjacent molecule, shapes active sites suitable for short malto-oligosaccharides, but hinders long stretches of oligosaccharides. The conserved residue of E44 protrudes at subsite +2, elucidating the hydrolysis pattern of the substrate by the glucose unit from the reducing end. The structural analysis provides a molecular basis for the substrate specificity and the enzymatic property, and has potential industrial application for protein engineering.

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are secreted by Gram-negative bacteria and function as primary virulence-promoting macromolecules that deliver multiple cytopathic and cytotoxic effector domains into the host cytoplasm. Among these effectors, Ras/Rap1-specific endopeptidase (RRSP) catalyzes the sequence-specific cleavage of the Switch I region of the cellular substrates Ras and Rap1 that are crucial for host innate immune defenses during infection. To dissect the molecular basis underpinning RRSP-mediated substrate inactivation, we determined the crystal structure of an RRSP from the sepsis-causing bacterial pathogen Vibrio vulnificus (VvRRSP). Structural and biochemical analyses revealed that VvRRSP is a metal-independent TIKI family endopeptidase composed of an N-terminal membrane-localization and substrate-recruitment domain (N lobe) connected via an inter-lobe linker to the C-terminal active site–coordinating core β-sheet–containing domain (C lobe). Structure-based mutagenesis identified the 2His/2Glu catalytic residues in the core catalytic domain that are shared with other TIKI family enzymes and that are essential for Ras processing. In vitro KRas cleavage assays disclosed that deleting the N lobe in VvRRSP causes complete loss of enzymatic activity. Endogenous Ras cleavage assays combined with confocal microscopy analysis of HEK293T cells indicated that the N lobe functions both in membrane localization via the first α-helix and in substrate assimilation by altering the functional conformation of the C lobe to facilitate recruitment of cellular substrates. Collectively, these results indicate that RRSP is a critical virulence factor that robustly inactivates Ras and Rap1 and augments the pathogenicity of invading bacteria via the combined effects of its N and C lobes.

SummaryThe CRISPR-Cas system is the RNA-guided immune defense mechanism in bacteria and archaea. Csm1 belongs to the Cas10 family, which is the common signature protein of the type III system. Csm1 is the largest subunit of the Csm interference complex in the type III-A subtype, which targets foreign DNA or RNA. Here, we report crystallographic and biochemical analyses of Thermococcus onnurineus Csm1, revealing a five-domain organization and single-stranded DNA (ssDNA)-specific nuclease activity associated with the N-terminal HD domain. This domain folds into permuted secondary structures in comparison with the HD domain of Cas3 and contains all the catalytically important residues. It exhibited both endo- and exonuclease activities in an Ni2+ or Mn2+-dependent manner. The narrow width of the active-site cleft appears to restrict the substrate specificity to ssDNA and thus to prevent Csm1 from cleaving double-stranded chromosomal DNA. These data suggest that Csm1 may function in DNA interference by the Csm effector complex.

SummaryRobust immune responses are essential for eliminating pathogens but must be metered to avoid prolonged immune activation and potential host damage. Upon recognition of microbial DNA, the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS) produces the second messenger cGAMP to initiate the stimulator of interferon genes (STING) pathway and subsequent interferon (IFN) production. We report that the direct interaction between cGAS and the Beclin-1 autophagy protein not only suppresses cGAMP synthesis to halt IFN production upon double-stranded DNA (dsDNA) stimulation or herpes simplex virus-1 infection, but also enhances autophagy-mediated degradation of cytosolic pathogen DNA to prevent excessive cGAS activation and persistent immune stimulation. Specifically, this interaction releases Rubicon, a negative autophagy regulator, from the Beclin-1 complex, activating phosphatidylinositol 3-kinase class III activity and thereby inducing autophagy to remove cytosolic pathogen DNA. Thus, the cGAS-Beclin-1 interaction shapes innate immune responses by regulating both cGAMP production and autophagy, resulting in well-balanced antimicrobial immune responses.

Protrudin is a FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain-containing protein involved in transport of neuronal cargoes and implicated in the onset of hereditary spastic paraplegia. Our image-based screening of the lipid binding domain library revealed novel plasma membrane localization of the FYVE domain of protrudin unlike canonical FYVE domains that are localized to early endosomes. The membrane binding study by surface plasmon resonance analysis showed that this FYVE domain preferentially binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) unlike canonical FYVE domains that specifically bind phosphatidylinositol 3-phosphate (PtdIns(3)P). Furthermore, we found that these phosphoinositides (PtdInsP) differentially regulate shuttling of protrudin between endosomes and plasma membrane via its FYVE domain. Protrudin mutants with reduced PtdInsP-binding affinity failed to promote neurite outgrowth in primary cultured hippocampal neurons. These results suggest that novel PtdInsP selectivity of the protrudin-FYVE domain is critical for its cellular localization and its role in neurite outgrowth.

Staphylothermus marinus maltogenic amylase (SMMA) is a novel extreme thermophile maltogenic amylase with an optimal temperature of 100 °C, which hydrolyzes α-(1-4)-glycosyl linkages in cyclodextrins and in linear malto-oligosaccharides. This enzyme has a long N-terminal extension that is conserved among archaic hyperthermophilic amylases but is not found in other hydrolyzing enzymes from the glycoside hydrolase 13 family. The SMMA crystal structure revealed that the N-terminal extension forms an N' domain that is similar to carbohydrate-binding module 48, with the strand-loop-strand region forming a part of the substrate binding pocket with several aromatic residues, including Phe-95, Phe-96, and Tyr-99. A structural comparison with conventional cyclodextrin-hydrolyzing enzymes revealed a striking resemblance between the SMMA N' domain position and the dimeric N domain position in bacterial enzymes. This result suggests that extremophilic archaea that live at high temperatures may have adopted a novel domain arrangement that combines all of the substrate binding components within a monomeric subunit. The SMMA structure provides a molecular basis for the functional properties that are unique to hyperthermophile maltogenic amylases from archaea and that distinguish SMMA from moderate thermophilic or mesophilic bacterial enzymes.

Chromosomes are condensed in all forms of life. SMC-based condensins are the key mediators in this process, but their molecular mechanisms remain elusive. Two different condensin complexes have been identified in prokaryotic organisms: MukB-MukE-MukF and SMC-ScpA-ScpB. This review focuses on comparison between the two machineries based on structural, biochemical and other related information in the light of their structure and function.

Malonamidase E2 (MAE2) from Bradyrhizobium japonicum is an enzyme that hydrolyzes malonamate to malonate and has a Ser-cis-Ser-Lys catalytic triad at the active site. The crystal structures of wild type and mutant MAE2 exhibited that the guanido group of Arg-158 could be involved in the binding of malonamate in which the negative charge of the carboxyl group could destabilize a negatively charged transition-state oxyanion in the enzymatic reaction. In an attempt to elucidate the specific roles of Arg-158, site-directed mutants, R158Q, R158E, and R158K, were prepared (see Table 1). The crystal structure of R158Q determined at 2.2 Angstrom resolution showed that the guanido group of Arg-158 was important for the substrate binding with the marginal structural change upon the mutation. The k(cat) value of R158Q significantly decreased by over 1500-fold and the catalytic activity of R158E could not be detected. The k(cat) value of R158K was similar to that of the wild type with the K(m) value drastically increased by 100-fold, suggesting that Lys-158 of R158K can stabilize the negative charge of the carboxylate in the substrate to some extent and contribute to the stabilization of the transition-state oxyanion, but a single amine group of Lys-158 in R158K could not precisely anchor the carboxyl group of malonamate compared with the guanido group of Arg-158. Our kinetic and structural evidences demonstrate that Arg-158 in MAE2 should be critical to both binding the substrate and stabilizing the transition-state oxyanion for the catalytic reaction of MAE2.

RbsD is the only protein whose biochemical function is unknown among the six gene products of the rbs operon involved in the active transport of ribose. FucU, a paralogue of RbsD conserved from bacteria to human, is also the only protein whose function is unknown among the seven gene products of the l-fucose regulon. Here we report the crystal structures of Bacillus subtilis RbsD, which reveals a novel decameric toroidal assembly of the protein. Nuclear magnetic resonance and other studies on RbsD reveal that the intersubunit cleft of the protein binds specific forms of d-ribose, but it does not have an enzyme activity toward the sugar. Likewise, FucU binds l-fucose but lacks an enzyme activity toward this sugar. We conclude that RbsD and FucU are cytoplasmic sugar-binding proteins, a novel class of proteins whose functional role may lie in helping influx of the sugar substrates.

Amidase signature family enzymes, which are widespread in nature, contain a newly identified Ser-cisSer-Lys catalytic triad in which the peptide bond between Ser131 and the preceding residue Gly130 is in a cis configuration. In order to characterize the property of the novel triad, we have determined the structures of five mutant malonamidase E2 enzymes that contain a Cys-cisSer-Lys, Ser-cisAla-Lys, or Ser-cisSer-Ala triad or a substitution of Gly130 with alanine. Cysteine cannot replace the role of Ser155 due to a hyper-reactivity of the residue, which results in the modification of the cysteine to cysteinyl sulfinic acid, most likely inside the expression host cells. The lysine residue plays a structural as well as a catalytic role, since the substitution of the residue with alanine disrupts the active site structure completely. The two observations are in sharp contrast with the consequences of the corresponding substitutions in the classical Ser-His-Asp triad. Structural data on the mutant containing the Ser-cisAla-Lys triad convincingly suggest that Ser131 plays an analogous catalytic role as the histidine of the Ser-His-Asp triad. The unusual cis configuration of Ser131 appears essential for the precise contacts of this residue with the other triad residues, as indicated by the near invariance of the preceding glycine residue (Gly130), structural data on the G130A mutant, and by a modeling experiment. The data provide a deep understanding of the role of each residue of the new triad at the atomic level and demonstrate that the new triad is a catalytic device distinctively different from the classical triad or its variants.

SEDL is an evolutionarily highly conserved protein in eukaryotic organisms. Deletions or point mutations in the SEDL gene are responsible for the genetic disease spondyloepiphyseal dysplasia tarda (SEDT), an X-linked skeletal disorder. SEDL has been identified as a component of the transport protein particle (TRAPP), critically involved in endoplasmic reticulum-to-Golgi vesicle transport. Herein, we report the 2.4 A resolution structure of SEDL, which reveals an unexpected similarity to the structures of the N-terminal regulatory domain of two SNAREs, Ykt6p and Sec22b, despite no sequence homology to these proteins. The similarity and the presence of unusually many solvent-exposed apolar residues of SEDL suggest that it serves regulatory and/or adaptor functions through multiple protein-protein interactions. Of the four known missense mutations responsible for SEDT, three mutations (S73L, F83S, V130D) map to the protein interior, where the mutations would disrupt the structure, and the fourth (D47Y) on a surface at which the mutation may abrogate functional interactions with a partner protein.

Over 20 enzymes denoted as cyclomaltodextrinase, maltogenic amylase, or neopullulanase that share 40-86% sequence identity with each other are found in public data bases. These enzymes are distinguished from typical alpha-amylases by containing a novel N-terminal domain and exhibiting preferential substrate specificities for cyclomaltodextrins (CDs) over starch. In this research field, a great deal of confusion exists regarding the features distinguishing the three groups of enzymes from one another. Although a different enzyme code has been assigned to each of the three different enzyme names, even a single differentiating enzymatic property has not been documented in the literature. On the other hand, an outstanding question related to this issue concerns the structural basis for the preference of these enzymes for CDs. To clarify the confusion and to address this question, we have determined the structures of two enzymes, one from alkalophilic Bacillus sp. I-5 and named cyclomaltodextrinase and the other from a Thermus species and named maltogenic amylase. The structure of the Bacillus enzyme reveals a dodecameric assembly composed of six copies of the dimer, which is the structural and functional unit of the Thermus enzyme and an enzyme named neopullulanase. The structure of the Thermus enzyme in complex with beta-CD led to the conclusion that Trp47, a well conserved N-terminal domain residue, contributes greatly to the preference for beta-CD. The common dimer formation through the novel N-terminal domain, which contributes to the preference for CDs by lining the active-site cavity, convincingly indicates that the three groups of enzymes are not different enough to preserve the different names and enzyme codes.

Both d - and l -isomers of N -(hydroxyaminocarbonyl)phenylalanine ( 1 ) were shown to have strong binding affinity towards carboxypeptidase A (CPA) with d - 1 being more potent than its enantiomer by 3-fold (Chung, S. J.; Kim, D. H. Bioorg. Med. Chem. 2001 , 9 , 185.). In order to understand the reversed stereochemical preference shown in the CPA inhibition, we have solved the crystal structures of CPA complexed with each enantiometer of 1 up to 1.75 A resolution. Inhibitor l - 1 whose stereochemistry belongs to the stereochemical series of substrate binds CPA like substrate does with its carbonyl oxygen coordinating to the active site zinc ion. Its hydroxyl is engaged in hydrogen bonding with the carboxylate of Glu-270. On the other hand, in binding of d - 1 to CPA, its terminal hydroxyl group is involved in interactions with the active site zinc ion and the carboxylate of Glu-270. In both CPA· 1 complexes, the phenyl ring in 1 is fitted in the substrate recognition pocket at the S 1 ′ subsite, and the carboxylate of the inhibitors forms bifurcated hydrogen bonds with the guanidinium moiety of Arg-145 and a hydrogen bond with the guanidinium of Arg-127. In the complex of CPA· d - 1 , the carboxylate of the inhibitor is engaged in hydrogen bonding with the phenolic hydroxyl of the down-positioned Tyr-248. While the l - 1 binding induces a concerted movement of the backbone amino acid residues at the active site, only the downward movement of Tyr-248 was noted when d - 1 binds to CPA.

Summary Multi-subunit SMC complexes control chromosome superstructure and promote chromosome disjunction, conceivably by actively translocating along DNA double helices. SMC subunits comprise an ABC ATPase “head” and a “hinge” dimerization domain connected by a 49 nm coiled-coil “arm.” The heads undergo ATP-dependent engagement and disengagement to drive SMC action on the chromosome. Here, we elucidate the architecture of prokaryotic Smc dimers by high-throughput cysteine cross-linking and crystallography. Co-alignment of the Smc arms tightly closes the interarm space and misaligns the Smc head domains at the end of the rod by close apposition of their ABC signature motifs. Sandwiching of ATP molecules between Smc heads requires them to substantially tilt and translate relative to each other, thereby opening up the Smc arms. We show that this mechanochemical gating reaction regulates chromosome targeting and propose a mechanism for DNA translocation based on the merging of DNA loops upon closure of Smc arms., Graphical Abstract, Highlights • Crystallography and in vivo cross-linking reveal the architecture of prokaryotic Smc • Juxtaposition of the Smc arms misaligns the two Smc ATPase domains • Smc head engagement mechanically opens an interarm space • A model for DNA loop extrusion driven by the SMC ATPase cycle is presented, By combining high-throughput in vivo cysteine cross-linking and crystallography, Diebold-Durand et al. construct a high-resolution model of full-length prokaryotic Smc. It reveals that the rod-shaped Smc dimer lacks chambers for DNA and features misaligned head domains. Smc head engagement mechanically opens an interarm space.

Background: Phytases hydrolyze phytic acid ( myo -inositol-hexakisphosphate) to less-phosphorylated myo -inositol derivatives and inorganic phosphate. Phytases are used in animal feed to reduce phosphate pollution in the environment. Recently, a thermostable, calcium-dependent Bacillus phytase was identified that represents the first example of the β propeller fold exhibiting phosphatase activity. We sought to delineate the catalytic mechanism and property of this enzyme. Results: The crystal structure of the enzyme in complex with inorganic phosphate reveals that two phosphates and four calcium ions are tightly bound at the active site. Mutation of the residues involved in the calcium chelation results in severe defects in the enzyme's activity. One phosphate ion, chelating all of the four calcium ions, is close to a water molecule bridging two of the bound calcium ions. Fluoride ion, which is expected to replace this water molecule, is an uncompetitive inhibitor of the enzyme. The enzyme is able to hydrolyze any of the six phosphate groups of phytate. Conclusions: The enzyme reaction is likely to proceed through a direct attack of the metal-bridging water molecule on the phosphorous atom of a substrate and the subsequent stabilization of the pentavalent transition state by the bound calcium ions. The enzyme has two phosphate binding sites, the "cleavage site", which is responsible for the hydrolysis of a substrate, and the "affinity site", which increases the binding affinity for substrates containing adjacent phosphate groups. The existence of the two nonequivalent phosphate binding sites explains the puzzling formation of the alternately dephosphorylated myo -inositol triphosphates from phytate and the hydrolysis of myo -inositol monophosphates.

Simmondsin, a material related to food intake inhibition from jojoba (Simmondsia chinensis), was transglycosylated by Bacillus stearothermophilus maltogenic amylase (BSMA) reaction with acarbose to synthesize an antiobese compound with hypoglycemic activity. Ten percent each of acarbose and simmondsin were mixed and incubated with BSMA at 55°C. Glycosylation products of simmondsin were observed by thin layer chromatography (TLC) and high performance ion chromatography (HPIC). The major transfer product was purified by using Biogel P-2 column. The structure was determined by matrix-assisted laser desorption ionization with time of flight (MALDI-TOF):mass spectrometry (MS) and 13 C-NMR. The major transglycosylation product was pseudotrisaccharide (PTS)-simmondsin, in which PTS was attached by an a-(1i6) glycosidic linkage to simmondsin. The administration of transglycosylated simmondsin with acarbose (200 mg:kg per day for 6 days) significantly reduced the food intake by 74%, comparable to 62% of simmondsin versus control in ob:ob mice. The transfer product (10 mg:kg) significantly suppressed the postprandial blood glucose response to starch (2 g:kg) by 68%, comparable to 60% of acarbose in Zucker fa:fa rats. The results indicated that the transfer products would be effective agents in lowering both food intake and blood glucose. © 2000 Elsevier Science B.V. All rights reserved.

Delta(5)-3-Ketosteroid isomerase from Pseudomonas testosteroni has been intensively studied as a prototype to understand an enzyme-catalyzed allylic isomerization. Asp(38) (pK(a) approximately 4.7) was identified as the general base abstracting the steroid C4beta proton (pK(a) approximately 12.7) to form a dienolate intermediate. A key and common enigmatic issue involved in the proton abstraction is the question of how the energy required for the unfavorable proton transfer can be provided at the active site of the enzyme and/or how the thermodynamic barrier can be drastically reduced. Answering this question has been hindered by the existence of two differently proposed enzyme reaction mechanisms. The 2.26 A crystal structure of the enzyme in complex with a reaction intermediate analogue equilenin reveals clearly that both the Tyr(14) OH and Asp(99) COOH provide direct hydrogen bonds to the oxyanion of equilenin. The result negates the catalytic dyad mechanism in which Asp(99) donates the hydrogen bond to Tyr(14), which in turn is hydrogen bonded to the steroid. A theoretical calculation also favors the doubly hydrogen-bonded system over the dyad system. Proton nuclear magnetic resonance analyses of several mutant enzymes indicate that the Tyr(14) OH forms a low barrier hydrogen bond with the dienolic oxyanion of the intermediate.

Electrospray ionization mass spectrometry (ESI-MS) is employed to directly analyze the limited trypsinolysis products of wild-type tumor necrosis factor-alpha (wtTNF-alpha) and its mutant, M3S. To determine the charge numbers of peaks of relatively small peptides in the ESI mass spectrum of a digest, a series of sodium-adduct ion peaks of each peptide are generated by adding a small quantity of NaCl to the digest before taking the spectrum. From the monitoring of the composition of proteolytic mixture as the incubation time is lengthened, it has been learned that the proteolysis of wtTNF-alpha by trypsin occurs sequentially: Arg2, Arg6, Arg32, Arg31, and Arg44, and that M3S is strongly resistant to the proteolysis. Since the cleavage sequence of wtTNF-alpha and the mutation-induced resistance of M3S are consistent with the structural features of the proteins, we can suggest a mutant more resistant to proteolysis than M3S, which has an additional point mutation, Ala35Leu or Ala35Ile.

Crystal structure of RNA helicase domain from genotype 1b hepatitis C virus has been determined at 2.3 A resolution by the multiple isomorphous replacement method. The structure consists of three domains that form a Y-shaped molecule. One is a NTPase domain containing two highly conserved NTP binding motifs. Another is an RNA binding domain containing a conserved RNA binding motif. The third is a helical domain that contains no β-strand. The RNA binding domain of the molecule is distinctively separated from the other two domains forming an interdomain cleft into which single stranded RNA can be modeled. A channel is found between a pair of symmetry-related molecules which exhibit the most extensive crystal packing interactions. A stretch of single stranded RNA can be modeled with electrostatic complementarity into the interdomain cleft and continuously through the channel. These observations suggest that some form of this dimer is likely to be the functional form that unwinds double stranded RNA processively by passing one strand of RNA through the channel and passing the other strand outside of the dimer. A “descending molecular see-saw” model is proposed that is consistent with directionality of unwinding and other physicochemical properties of RNA helicases.

A human tumor necrosis factor-alpha (TNF-alpha) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-A resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the N-terminal seven amino acids that is disordered in the structure of wild-type TNF-alpha. Compared with wild-type TNF-alpha, it exhibits 11- and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect and in vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-alpha. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29-36 into a rigid segment as a consequence of induced formation of intra- and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe56 projects into a hydrophobic cavity, while Ile52 is exposed to the bulk solvent. Ile52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-alpha. It is also expected that the 1.8-A resolution structure will serve as an accurate model for explaining the structure-function relationship of wild-type TNF-alpha and many TNF-alpha mutants reported previously and for the design of new TNF-alpha mutants.

The periplasmic histidine-binding protein, HisJ, is a receptor for the histidine permease of Salmonella typhimurium. Receptors of this type are composed of two lobes that are far apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation). The binding of the ligand, in a cleft between the lobes, stabilizes the closed conformation. Such receptors have several functions in transport: interaction with the membrane-bound complex, transmission of a transmembrane signal to hydrolyze ATP, and receiving a signal to open the lobes and release the ligand. In this study the mechanism of action of HisJ was further investigated using mutant proteins defective in ligand binding activity and closed form-specific monoclonal antibodies (Wolf, A., Shaw, E. W., Nikaido, K., and Ames G. F.-L.(1994) J. Biol. Chem. 269, 23051-23058). Y14H is defective in stabilization of the closed form, does not assume the closed empty form, and assumes an altered closed liganded form. T121A and G119R are similar to Y14H, but assume a normal closed liganded form. S72P binds the ligand to the open form, but does not assume a recognizable closed form. S92F is defective in the ability to undergo conformational change and to stabilize the closed form. All other mutant proteins appear to fall within one of these four categories. The biochemical characterization of these mutant proteins agrees with the structural analysis of the protein. We suggest that mutant proteins that do not assume the normal closed form, in addition to their defect in ligand binding, fail to interact with the membrane-bound complex and/or to transmit transmembrane signals.

The substrate-binding site of a protein with multiple specificity should satisfy geometric and energetic complementarity for several different substrates. The structural basis of the multiple ligand specificity of the periplasmic lysine-, arginine-, ornithine-binding protein (LAO) was investigated by determining and analyzing the structures of the protein unliganded and liganded with each of the three high-affinity ligands (L-lysine, L-arginine, and L-ornithine) and with one low-affinity ligand (L-histidine). The geometric complementarity is achieved primarily by virtue of the large size of the ligand-binding site which can accommodate the maximum common volume of the four ligands plus three water molecules. The optimization of energetic complementarity is achieved by the relocation of protein-bound water molecules and by the movement of the Asp-11 side chain. The structure of the LAO-histidine complex indicates that the 30-fold reduced affinity of the protein for histidine is primarily due to unavailability of one ionic interaction of the histidine side chain with the protein which is present in the other three complexes.

Bacterial periplasmic binding proteins are initial receptors in the process of active transport across cell membranes and/or chemotaxis. Among them, the histidine-binding protein (HisJ) has been extensively studied from the biochemical, physiological, and genetic points of view. The three-dimensional crystal structure of the histidine-binding protein complexed with histidine has been determined at 2.5-A resolution by the molecular replacement method using a probe structure the previously solved lysine-liganded structure of the lysine-, arginine-, ornithine-binding protein (LAO), which shares 70% sequence identity with HisJ. The structure is bi-lobate; the two lobes, one bigger than the other, are connected by two short strands and are in contact with each other (closed) enclosing the histidine. Charged, polar, and non-polar side chains, as well as the peptide backbone, are involved in tight binding of the histidine. The bound histidine is involved in eight direct hydrogen bonds, six with the bigger lobe and two with the smaller lobe, in one potential water-mediated hydrogen bond with the bigger lobe, as well as in ionic interactions. The HisJ residues surrounding the ligand are the same as the LAO residues interacting with lysine, except for residue 52 which is leucine in HisJ and phenylalanine in LAO. The Leu-52 in HisJ makes a hydrophobic interaction with the imidazole ring of histidine. Of seven mutations affecting the ligand-binding site, five are located in the ligand-binding site, one in a connecting strand, and one at the domains interface. Based on comparisons among related binding proteins, the specific interactions between the ligands and the respective binding protein residues are predicted for the glutamine-binding protein and the opines-binding protein.

Many proteins exhibit a large-scale movement of rigid globular domains. Among these, bacterial periplasmic binding proteins involved in substrate transport, or transport and chemotaxis, can be used as prototypes for understanding the mechanism of the movement. Such movements have been found to be associated with specific functions, such as substrate binding, catalysis, and recognition by other biomolecules. We have determined the three-dimensional structures of the lysine/arginine/ornithine-binding protein (LAO) from Salmonella typhimurium with and without lysine by x-ray crystallographic methods at 1.8- and 1.9-A resolution, respectively. The structures are composed of two lobes held together by two short connecting strands. The two lobes are far apart in the unliganded structure, but in contact with each other in the lysine-liganded structure. The large movement of the lobes is a consequence of a 52 degrees rotation of a single backbone torsion angle in the first connecting strand and of distributed smaller changes of three backbone torsion angles of the second connecting strand. The absence of contact between the lysine and the connecting strands suggests that the ligand does not induce the conformational change directly. We instead propose that the unliganded protein undergoes a dynamic change between an "open" and a "closed" conformation and that the role of the ligand is to stabilize the closed conformation. We discuss the nature of a surface area which might be recognized by the membrane-bound complex of these amino acids transport systems.