Structure of D-allose binding protein from Escherichia coli bound to D-allose at 1.8 A resolution

ABC transport systems for import or export of nutrients and other substances across the cell membrane are widely distributed in nature. In most bacterial systems, a periplasmic component is the primary determinant of specificity of the transport complex as a whole. We report here the crystal structure of the periplasmic binding protein for the allose system (ALBP) from Escherichia coli, solved at 1.8 A resolution using the molecular replacement method. As in the other members of the family (especially the ribose binding protein, RBP, with which it shares 35 % sequence homology), this structure consists of two similar domains joined by a three-stranded hinge region. The protein is believed to exist in a dynamic equilibrium of closed and open conformations in solution which is an important part of its function. In the closed ligand-bound form observed here, D-allose is buried at the domain interface. Only the beta-anomer of allopyranose is seen in the crystal structure, although the alpha-anomer can potentially bind with a similar affinity. Details of the ligand-binding cleft reveal the features that determine substrate specificity. Extensive hydrogen bonding as well as hydrophobic interactions are found to be important. Altogether ten residues from both the domains form 14 hydrogen bonds with the sugar. In addition, three aromatic rings, one from each domain with faces parallel to the plane of the sugar ring and a third perpendicular, make up a hydrophobic stacking surface for the ring hydrogen atoms. Our results indicate that the aromatic rings forming the sugar binding cleft can sterically block the binding of any hexose epimer except D-allose, 6-deoxy-allose or 3-deoxy-glucose; the latter two are expected to bind with reduced affinity, due to the loss of some hydrogen bonds. The pyranose form of the pentose, D-ribose, can also fit into the ALBP binding cleft, although with lower binding affinity. Thus, ALBP can function as a low affinity transporter for D-ribose. The significance of these results is discussed in the context of the function of allose and ribose transport systems.     

Structure of ricin B-chain at 2.5 A resolution

The heterodimeric plant toxin ricin has been refined to 2.5 A resolution. The B-chain lectin (RTB) is described in detail. The protein has two major domains, each of which has a galactose binding site. RTB has no regular secondary structure but displays several omega loops. Each RTB domain is made of three copies of a primitive 40 residue folding unit, which pack around a pseudo threefold axis. In each domain, galactose binds in a shallow cleft formed by a three residue peptide kink on the bottom and an aromatic ring on the top. At the back of the cleft, an aspartate forms hydrogen bonds to the C3 and C4 hydroxyls of galactose, whereas a glutamine bonds to the C4 alcohol, helping to define specific epimer binding. In addition to analyzing the sugar binding mechanism, the assembly of subdomain units around the pseudo threefold axis of each domain is described. The subdomains contribute conserved Trp, Leu, and Ile residues to a compact central hydrophobic core. This tight threefold binding probably drives the peptide folding and stabilizes the protein structure.     

Ribose and glucose-galactose receptors. Competitors in bacterial chemotaxis.

The periplasmic ribose and glucose-galactose receptors (binding proteins) of Gram-negative bacteria compete for a common inner membrane receptor in bacterial chemotaxis, as well as being the essential primary receptors for their respective membrane transport systems. The high-resolution structures of the periplasmic receptors for ribose (from Escherichia coli) and glucose or galactose (from both Salmonella typhimurium and E. coli) are compared here to outline some features that may be important in their dual functions. The overall structure of each protein consists of two similar domains, both of which are made up of two non-contiguous segments of amino acid chain. Each domain is composed of a core of beta-sheet flanked on both sides with alpha-helices. The two domains are related to each other by an almost perfect intramolecular axis of symmetry. The ribose receptor is smaller as a result of a number of deletions in its sequence relative to the glucose-galactose receptor, mostly occurring in the loop regions; as a result, this protein is also more symmetrical. Many structural features, including some hydrophobic core interactions, a buried aspartate residue and several unusual turns, are conserved between the two proteins. The binding sites for ligand are in similar locations, and built along similar principles, although none of the specific interactions with the sugars is conserved. A comparison shows further that slightly different rotations relate the domains to each other in the three proteins, with the ribose receptor being the most closed, and the Salmonella glucose-galactose receptor the most open. The primary axis of relative rotation is almost perpendicular to that which describes the intramolecular symmetry in each case. These relative rotations of the domains are accompanied by the sliding of some helices as the structures adjust themselves to relieve strain. The hinges which are responsible for most of these relative domain rotations are very similar in the three proteins, consisting of a symmetrical arrangement of beta-strands and alpha-helices and two conserved water molecules that are critical to the hydrogen bonding in the important interdomain region. A region of high sequence and structural similarity between the ribose and glucose-galactose receptors is also located around the intramolecular symmetry axis, on the opposite side of the proteins from the hinge region. This region is that which is altered most by the relative rotations, and is the location of most of the known mutations which affect chemotaxis and transport in the ribose receptor.     

The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A

We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis. In vivo, this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli. It consists of two similar lobes, each formed by a parallel beta-sheet flanked by alpha-helices on both sides. Both are connected by a hinge region consisting of two antiparallel beta-strands and an alpha-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.     

Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.     

Ice-binding mechanism of winter flounder antifreeze proteins

We have studied the winter flounder antifreeze protein (AFP) and two of its mutants using molecular dynamics simulation techniques. The simulations were performed under four conditions: in the gas phase, solvated by water, adsorbed on the ice (2021) crystal plane in the gas phase and in aqueous solution. This study provided details of the ice-binding pattern of the winter flounder AFP. Simulation results indicated that the Asp, Asn, and Thr residues in the AFP are important in ice binding and that Asn and Thr as a group bind cooperatively to the ice surface. These ice-binding residues can be collected into four distinct ice-binding regions: Asp-1/Thr-2/Asp-5, Thr-13/Asn-16, Thr-24/Asn-27, and Thr-35/Arg-37. These four regions are 11 residues apart and the repeat distance between them matches the ice lattice constant along the (1102) direction. This match is crucial to ensure that all four groups can interact with the ice surface simultaneously, thereby, enhancing ice binding. These Asx (x = p or n)/Thr regions each form 5-6 hydrogen bonds with the ice surface: Asn forms about three hydrogen bonds with ice molecules located in the step region while Thr forms one to two hydrogen bonds with the ice molecules in the ridge of the (2021) crystal plane. Both the distance between Thr and Asn and the ordering of the two residues are crucial for effective ice binding. The proper sequence is necessary to generate a binding surface that is compatible with the ice surface topology, thus providing a perfect "host/guest" interaction that simultaneously satisfies both hydrogen bonding and van der Waals interactions. The results also show the relation among binding energy, the number of hydrogen bonds, and the activity. The activity is correlated to the binding energy, and in the case of the mutants we have studied the number of hydrogen bonds. The greater the number of the hydrogen bonds the greater the antifreeze activity. The roles van der Waals interactions and the hydrophobic effect play in ice binding are also highlighted. For the latter it is demonstrated that the surface of ice has a clathratelike structure which favors the partitioning of hydrophobic groups to the surface of ice. It is suggested that mutations that involve the deletion of hydrophobic residues (e.g., the Leu residues) will provide insight into the role the hydrophobic effect plays in partitioning these peptides to the surface of ice.     

Structure of a triclinic ternary complex of horse liver alcohol dehydrogenase at 2.9 Å resolution

The structure of a triclinic complex between liver alcohol dehydrogenase, reduced coenzyme NADH, and the inhibitor dimethylsulfoxide has been determined to 2.9 Å resolution using isomorphous replacement methods. The heavy-atom positions were derived by molecular replacement methods using phase angles derived from a model of the orthorhombic apoenzyme structure previously determined to 2.4 Å resolution. A model of the present holoenzyme molecule was built on a Vector General 3400 display system using the RING system of programs. This model gave a crystallographic R-value of 37.9%.There are extensive conformational differences between the protein molecules in the two forms. The conformational change involves a rotation of 7.5 ° of the catalytic domains relative to the coenzyme binding domains. A hinge region for this rotation is defined within a hydrophobic core between two helices. The internal structures of the domains are preserved with the exception of a movement of a small loop in the coenzyme binding domain. A cleft between the domains is closed by this coenzyme-induced conformational change, making the active site less accessible from solution and thus more hydrophobic.The two crystallographically independent subunits are very similar and bind both coenzyme and inhibitor in an identical way within the present limits of error. The coenzyme molecule is bound in an extended conformation with the two ends in hydrophobic crevices on opposite sides of the central pleated sheet of the coenzyme binding domain. There are hydrogen bonds to oxygen atoms of the ribose moities from Asp223, Lys228 and His51. The pyrophosphate group is in contact with the side-chains of Arg47 and Arg369.No new residues are brought into the active site compared to the apoenzyme structure. The active site zinc atom is close to the hinge region, where the smallest structural changes occur. Small differences in the co-ordination geometry of the ligands Cys46, His67 and Cysl74 are not excluded and may account for the ordered mechanism. The oxygen atom of the inhibitor dimethylsulfoxide is bound directly to zinc confirming the structural basis for the suggested mechanism of action based on studies of the apoenzyme structure.     

Molecular dynamics simulations of a beta-hairpin fragment of protein G: balance between side-chain and backbone forces

How is the native structure encoded in the amino acid sequence? For the traditional backbone centric view, the dominant forces are hydrogen bonds (backbone) and phi-psi propensity. The role of hydrophobicity is non-specific. For the side-chain centric view, the dominant force of protein folding is hydrophobicity. In order to understand the balance between backbone and side-chain forces, we have studied the contributions of three components of a beta-hairpin peptide: turn, backbone hydrogen bonding and side-chain interactions, of a 16-residue fragment of protein G. The peptide folds rapidly and cooperatively to a conformation with a defined secondary structure and a packed hydrophobic cluster of aromatic side-chains. Our strategy is to observe the structural stability of the beta-hairpin under systematic perturbations of the turn region, backbone hydrogen bonds and the hydrophobic core formed by the side-chains, respectively. In our molecular dynamics simulations, the peptides are solvated. with explicit water molecules, and an all-atom force field (CFF91) is used. Starting from the original peptide (G41EWTYDDATKTFTVTE56), we carried out the following MD simulations. (1) unfolding at 350 K; (2) forcing the distance between the C(alpha) atoms of ASP47 and LYS50 to be 8 A; (3) deleting two turn residues (Ala48 and Thr49) to form a beta-sheet complex of two short peptides, GEWTYDD and KTFTVTE; (4) four hydrophobic residues (W43, Y45, F52 and T53) are replaced by a glycine residue step-by-step; and (5) most importantly, four amide hydrogen atoms (T44, D46, T53, and T55, which are crucial for backbone hydrogen bonding), are substituted by fluorine atoms. The fluorination not only makes it impossible to form attractive hydrogen bonding between the two beta-hairpin strands, but also introduces a repulsive force between the two strands due to the negative charges on the fluorine and oxygen atoms. Throughout all simulations, we observe that backbone hydrogen bonds are very sensitive to the perturbations and are easily broken. In contrast, the hydrophobic core survives most perturbations. In the decisive test of fluorination, the fluorinated peptide remains folded under our simulation conditions (5 ns, 278 K). Hydrophobic interactions keep the peptide folded, even with a repulsive force between the beta-strands. Thus, our results strongly support a side-chain centric view for protein folding.     

Direct analysis of backbone-backbone hydrogen bond formation in protein folding transition states

Here we investigate the role of backbone-backbone hydrogen bonding interactions in stabilizing the protein folding transition states of two model protein systems, the B1 domain of protein L (ProtL) and the P22 Arc repressor. A backbone modified analogue of ProtL containing an amide-to-ester bond substitution between residues 105 and 106 was prepared by total chemical synthesis, and the thermodynamic and kinetic parameters associated with its folding reaction were evaluated. Ultimately, these parameters were used in a Phi-value analysis to determine if the native backbone-backbone hydrogen bonding interaction perturbed in this analogue (i.e. a hydrogen bond in the first beta-turn of ProtL's beta-beta-alpha-beta-beta fold) was formed in the transition state of ProtL's folding reaction. Also determined were the kinetic parameters associated with the folding reactions of two Arc repressor analogues, each containing an amide-to-ester bond substitution in the backbone of their polypeptide chains. These parameters were used together with previously established thermodynamic parameters for the folding of these analogues in Phi-value analyses to determine if the native backbone-backbone hydrogen bonding interactions perturbed in these analogues (i.e. a hydrogen bond at the end of the intersubunit beta-sheet interface and hydrogen bonds at the beginning of the second alpha-helix in Arc repressor's beta-alpha-alpha structure) were formed in the transition state of Arc repressor's folding reaction. Our results reveal that backbone-backbone hydrogen bonding interactions are formed in the beta-turn and alpha-helical transition state structures of ProtL and Arc repressor, respectively; and they were not formed in the intersubunit beta-sheet interface of Arc repressor, a region of Arc repressor's polypeptide chain previously shown to have other non-native-like conformations in Arc's protein folding transition state.     

Desolvation shell of hydrogen bonds in folded proteins,protein complexes and folding pathways

A few backbone hydrogen bonds (HBS) in native protein folds are poorly protected from water attack: their desolvation shell contains an inordinately low number of hydrophobic residues. Thus, an approach by solvent-structuring moieties of a binding partner should contribute significantly to enhance their stability. This effect represents an important factor in the site specificity inherent to protein binding, as inferred from a strong correlation between poorly desolvated HBs and binding sites. The desolvation shells were also examined in a dynamic context: except for a few singular under-protected bonds, the size of desolvation shells is preserved along the folding trajectory.     

Mutational analysis and NMR spectroscopy of quail cysteine and glycine-rich protein CRP2 reveal an intrinsic segmental flexibility of LIM domains.

The LIM domain is a conserved cysteine and histidine-containing structural module of two tandemly arranged zinc fingers. It has been identified in single or multiple copies in a variety of regulatory proteins, either in combination with defined functional domains, like homeodomains, or alone, like in the CRP family of LIM proteins. Structural studies of CRP proteins have allowed a detailed evaluation of interactions in LIM-domains at the molecular level. The packing interactions in the hydrophobic core have been identified as a significant contribution to the LIM domain fold, whereas hydrogen bonding within each single zinc binding site stabilizes zinc finger geometry in a so-called "outer" or "indirect" coordination sphere. Here we report the solution structure of a point-mutant of the carboxyl-terminal LIM domain of quail cysteine and glycine-rich protein CRP2, CRP2(LIM2)R122A, and discuss the structural consequences of the disruption of the hydrogen bond formed between the guanidinium side-chain of Arg122 and the zinc-coordinating cysteine thiolate group in the CCHC rubredoxin-knuckle. The structural analysis revealed that the three-dimensional structure of the CCHC zinc binding site in CRP2(LIM2)R122A is adapted as a consequence of the modified hydrogen bonding pattern. Additionally, as a result of the conformational rearrangement of the zinc binding site, the packing interactions in the hydrophobic core region are altered, leading to a change in the relative orientation of the two zinc fingers with a concomitant change in the solvent accessibilities of hydrophobic residues located at the interface of the two modules. The backbone dynamics of residues located in the folded part of CRP2(LIM2)R122A have been characterized by proton-detected(15)N NMR spectroscopy. Analysis of the R2/R1ratios revealed a rotational correlation time of approximately 6.2 ns and tumbling with an axially symmetric diffusion tensor (D parallel/D perpendicular=1.43). The relaxation data were also analyzed using a reduced spectral density mapping approach. As in wild-type CRP2(LIM2), significant mobility on a picosecond/nanosecond time-scale was detected, and conformational exchange on a microsecond time-scale was identified for residues located in loop regions between secondary structure elements. In summary, the relative orientation of the two zinc binding sites and the accessibility of hydrophobic residues is not only determined by hydrophobic interactions, but can also be modified by the formation and/or breakage of hydrogen bonds. This may be important for the molecular interactions of an adaptor-type LIM domain protein in macromolecular complexes, particularly for the modulation of protein-protein interactions.     

Crystallographic structure and substrate-binding interactions of the molybdate-binding protein of the phytopathogen Xanthomonas axonopodis pv. citri

In Xanthomonas axonopodis pv. citri (Xac or X. citri), the modA gene codes for a periplasmic protein (ModA) that is capable of binding molybdate and tungstate as part of the ABC-type transporter required for the uptake of micronutrients. In this study, we report the crystallographic structure of the Xac ModA protein with bound molybdate. The Xac ModA structure is similar to orthologs with known three-dimensional structures and consists of two nearly symmetrical domains separated by a hinge region where the oxyanion-binding site lies. Phylogenetic analysis of different ModA orthologs based on sequence alignments revealed three groups of molybdate-binding proteins: bacterial phytopathogens, enterobacteria and soil bacteria. Even though the ModA orthologs are segregated into different groups, the ligand-binding hydrogen bonds are mostly conserved, except for Archaeglobus fulgidus ModA. A detailed discussion of hydrophobic interactions in the active site is presented and two new residues, Ala38 and Ser151, are shown to be part of the ligand-binding pocket.     

Forces stabilizing proteins

The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. (1) Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a –CH₂− group on folding contributes 1.1 ± 0.5 kcal/mol to protein stability. (2) The burial of non-polar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. (3) Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1 ± 0.8 kcal/mol to protein stability. (4) The contribution of hydrogen bonds to protein stability is strongly context dependent. (5) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (6) Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. (7) Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability.     

Structural evidence for a dominant role of nonpolar interactions in the binding of a transport/chemosensory receptor to its highly polar ligands.

The receptor, a maltose/maltooligosaccharide-binding protein, has been found to be an excellent system for the study of molecular recognition because its polar and nonpolar binding functions are segregated into two globular domains. The X-ray structures of the "closed" and "open" forms of the protein complexed with maltose and maltotetraitol have been determined. These sugars have approximately 3 times more accessible polar surface (from OH groups) than nonpolar surface (from small clusters of sugar ring CH bonds). In the closed structures, the oligosaccharides are buried in the groove between the two domains of the protein and bound by extensive hydrogen bonding interactions of the OH groups with the polar residues confined mostly in one domain and by nonpolar interactions of the CH clusters with four aromatic residues lodged in the other domain. Substantial contacts between the sugar hydroxyls and aromatic residues are also formed. In the open structures, the oligosaccharides are bound almost exclusively in the domain rich in aromatic residues. This finding, along with the analysis of buried surface area due to complex formations in the open and closed structures, supports a major role for nonpolar interactions in initial ligand binding even when the ligands have significantly greater potential for highly specific polar interactions.     

Energetics and cooperativity of tertiary hydrogen bonds in RNA structure.

Tertiary interactions that allow RNA to fold into intricate three-dimensional structures are being identified, but little is known about the thermodynamics of individual interactions. Here we quantify the tertiary structure contributions of individual hydrogen bonds in a "ribose zipper" motif of the recently crystallized Tetrahymena group I intron P4-P6 domain. The 2'-hydroxyls of P4-P6 nucleotides C109/A184 and A183/G110 participate in forming the "teeth" of the zipper. These four nucleotides were substituted in all combinations with their 2'-deoxy and (separately) 2'-methoxy analogues, and thermodynamic effects on the tertiary folding DeltaG degrees ' were assayed by the Mg2+ dependence of electrophoretic mobility in nondenaturing gels. The 2'-deoxy series showed a consistent trend with an average contribution to the tertiary folding DeltaG degrees' of -0.4 to -0.5 kcal/mol per hydrogen bond. Contributions were approximately additive, reflecting no cooperativity among the hydrogen bonds. Each "tooth" of the ribose zipper (comprising two hydrogen bonds) thus contributes about -1.0 kcal/mol to the tertiary folding DeltaG degrees'. Single 2'-methoxy substitutions destabilized folding by approximately 1 kcal/mol, but the trend reversed with multiple 2'-methoxy substitutions; the folding DeltaG degrees' for the quadruple 2'-methoxy derivative was approximately unchanged relative to wild-type. On the basis of these data and on temperature-gradient gel results, we conclude that entropically favorable hydrophobic interactions balance enthalpically unfavorable hydrogen bond deletions and steric clashes for multiple 2'-methoxy substitutions. Because many of the 2'-deoxy derivatives no longer have the characteristic hydrogen-bond patterns of the ribose zipper motif but simply have individual long-range ribose-base or ribose-ribose hydrogen bonds, we speculate that the energetic value of -0.4 to -0.5 kcal/mol per tertiary hydrogen bond may be more generally applicable to RNA folding.     

Insufficient hydrogen-bond desolvation and prion-related disease.

A structuring and eventual exclusion of water surrounding backbone hydrogen bonds takes place during protein folding as hydrophobic residues cluster around such bonds. Taken as an average over all hydrogen bonds, the extent of desolvation is nearly a constant of motion, as revealed by re-examination of the longest all-atom trajectory with explicit solvent [Y. Duan & P. A. Kollman (1998) Science 282, 740]. Furthermore, this extent of desolvation is preserved across native soluble proteins, except for cellular prion proteins. Thus, a physico-chemical picture of prion-related disease emerges. The epitope for protein-X binding, the region undergoing vast conformational change and the trigger and locker for this change are inferred from the location of under-desolvated hydrogen bonds in the cellular prion protein.     

Stability and sugar recognition ability of ricin-like carbohydrate binding domains

Lectins are a class of proteins known for their novel binding to saccharides. Understanding this sugar recognition process can be crucial in creating structure-based designs of proteins with various biological roles. We focus on the sugar binding of a particular lectin, ricin, which has two β-trefoil carbohydrate-binding domains (CRDs) found in several plant protein toxins. The binding ability of possible sites of ricin-like CRD has been puzzling. The apo and various (multiple) ligand-bound forms of the sugar-binding domains of ricin were studied by molecular dynamics simulations. By evaluating structural stability, hydrogen bond dynamics, flexibility, and binding energy, we obtained a detailed picture of the sugar recognition of the ricin-like CRD. Unlike what was previously believed, we found that the binding abilities of the two known sites are not independent of each other. The binding ability of one site is positively affected by the other site. While the mean positions of different binding scenarios are not altered significantly, the flexibility of the binding pockets visibly decreases upon multiple ligand binding. This change in flexibility seems to be the origin of the binding cooperativity. All the hydrogen bonds that are strong in the monoligand state are also strong in the double-ligand complex, although the stability is much higher in the latter form due to cooperativity. These strong hydrogen bonds in a monoligand state are deemed to be the essential hydrogen bonds. Furthermore, by examining the structural correlation matrix, the two domains are structurally one entity. Galactose hydroxyl groups, OH4 and OH3, are the most critical parts in both site 1α and site 2γ recognition.     

Point mutations in a hinge linking the small and large domains of beta-actin result in trapped folding intermediates bound to cytosolic chaperonin CCT

The 30-A cryo-EM-derived structure of apo-CCT-alpha-actin shows actin opened up across its nucleotide-binding cleft and binding to either of two CCT subunit pairs, CCTbeta-CCTdelta or CCTepsilon-CCTdelta, in a similar 1:4 arrangement. The two main duplicated domains of native actin are linked twice, topologically, by the connecting residues, Q137-S145 and P333-S338, and are tightly held together by hydrogen bonding with bound adenine nucleotide. We carried out a mutational screen to find residues in actin that might be involved in the huge rotations observed in the CCT-bound folding intermediate. When two evolutionarily highly conserved glycine residues of beta-actin, G146 and G150, were changed to proline, both mutant actin proteins were poorly processed by CCT in in vitro translation assays; they become arrested on CCT. A three-dimensional reconstruction of the substrate-bound ring of the apo-CCT-beta-actin complex shows that beta-actin G150P is not able to bind across the chaperonin cavity to interact with the CCTdelta subunit. beta-actin G150P seems tightly packed and apparently bound only to the CCTbeta and CCTepsilon subunits, which further indicates that these CCT subunits drive the interaction between CCT and actin. Hinge opening seems to be critical for actin folding, and we suggest that residues G146 and G150 are important components of the hinge around which the rigid subdomains, presumably already present in early actin folding intermediates, rotate during CCT-assisted folding.     

Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge

Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptor-binding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptor-bound) arrestin conformation is essentially the same in both wild type and mutant forms.