Gap junction channel structure in the early 21st century: facts and fantasies

Gap junction channels connect the cytoplasms of adjacent cells through the end-to-end docking of single-membrane structures called connexons, formed by a ring of six connexin monomers. Each monomer contains four transmembrane alpha-helices, for a total of 24 alpha-helices in a connexon. The fundamental structure of the connexon pore is probably similar in unpaired connexons and junctional channels, and for channels formed by different connexin isoforms. Nevertheless, variability in results from structurally focused mutagenesis and electrophysiological studies raise uncertainty about the specific assignments of the transmembrane helices. Mapping of human mutations onto a suggested C(alpha) model predicts that mutations that disrupt helix-helix packing impair channel function. An experimentally determined structure at atomic resolution will be essential to confirm and resolve these concepts.     

Secondary structure based analysis and classification of biological interfaces: identification of binding motifs in protein-protein interactions

MOTIVATION: The increasing amount of data on protein-protein interaction needs to be rationalized for deriving guidelines for the alteration or design of an interface between two proteins. RESULTS: We present a detaild structural analysis and comparison of homo- versus heterodimeric protein-protein interfaces. Regular secondary structures (helices and strands) are the main components of the former, whereas non-regular structures (turns, loops, etc.) frequently mediate interactions in the latter. Interface helices get longer with increasing interface area, but only in heterocomplexes. On average, the homodimers have longer helical segments and prominent helix-helix pairs. There is a surprising distinction in the relative orientation of interface helices, with a tendency for aligned packing in homodimers and a clear preference for packing at 90 degrees in heterodimers. Arg and the aromatic residues have a higher preference to occur in all secondary structural elements (SSEs) in the interface. Based on the dominant SSE, the interfaces have been grouped into four classes: alpha, beta, alphabeta and non-regular. Identity between protein and interface classes is the maximum for alpha proteins, but rather mediocre for the other protein classes. The interface classes of the two chains forming a heterodimer are often dissimilar. Eleven binding motifs can capture the prominent architectural features of most of the interfaces.     

The formation of a salt bridge between helices 3 and 6 is responsible for the constitutive activity and lack of hormone responsiveness of the naturally occurring L457R mutation of the human lutropin receptor

The human lutropin receptor (hLHR) plays a pivotal role in reproductive endocrinology. A number of naturally occurring mutations of the hLHR have been identified that cause the receptor to become constitutively active. To gain further insights into the structural basis for the activation of the hLHR by activating mutations, we chose to examine a particularly strong constitutively activating mutation of this receptor, L457R, in which a leucine that is highly conserved among rhodopsin-like G protein-coupled receptors in helix 3 has been substituted with arginine. Using both disruptive as well as reciprocal mutagenesis strategies, our studies demonstrate that the ability of L457R to stabilize an active form of the hLHR is because of the formation of a salt bridge between the replacing amino acid and Asp-578 in helix 6. Such a lock between the transmembrane portions of helices 3 and 6 is concurrent with weakening the connections between the cytosolic ends of the same helices, including the interaction found in the wild-type receptor between Arg-464, of the (E/D)R(Y/W) motif, and Asp-564. This structural effect is properly marked by the increase in the solvent accessibility of selected amino acids at the cytosolic interfaces between helices 3 and 6. The integrity of the conserved amino acids Asn-615 and Asn-619 in helix 7 is required for the transfer of the structural change from the activating mutation site to the cytosolic interface between helices 3 and 6. The results of in vitro and computational experiments further suggest that the structural trigger of the constitutive activity of the L457R mutant may also be responsible for its lack of hormone responsiveness.     

Structure-function analysis of the alpha5 and the alpha13 helices of human glucokinase: description of two novel activating mutations

It was recently described that the alpha5 and the alpha13 helices of human pancreatic glucokinase play a major role in the allosteric regulation of the enzyme. In order to understand the structural importance of these helices, we have performed site-directed mutagenesis to generate glucokinase derivatives with altered residues. We have analyzed the kinetic parameters of these mutated forms and compared them with wild-type and previously defined activating mutations in these helices (A456V and Y214C). We found two new activating mutations, A460R and Y215A, which increase the affinity of the enzyme for glucose. Our results suggest that substitutions in the alpha5 or the alpha13 helices that favor the closed, active conformation of the enzyme, either by improving the interaction with surrounding residues or by improving the flexibility of the region defined by these two helices, enhance the affinity of the enzyme for glucose, and therefore its performance as a glucose phosphorylating enzyme.     

Crystal structure of an aminoimidazole riboside kinase from Salmonella enterica: implications for the evolution of the ribokinase superfamily

The crystal structures of a Salmonella enterica aminoimidazole riboside (AIRs) kinase, its complex with the substrate AIRs, and its complex with AIRs and an ATP analog were determined at 2.6 angstroms, 2.9 angstroms, and 2.7 angstroms, respectively. The product of the Salmonella-specific gene stm4066, AIRs kinase, is a homodimer with one active site per monomer. The core structure, consisting of an eight-stranded beta sheet flanked by eight alpha helices, indicates that AIRs kinase is a member of the ribokinase superfamily. Unlike ribokinase and adenosine kinase in this superfamily, AIRs kinase does not show significant conformational changes upon substrate binding. The active site is covered by a lid formed by residues 16-28 and 86-100. A comparison of the structure of AIRs kinase with other ribokinase superfamily members suggests that the active site lid and conformational changes that occur upon substrate binding may be advanced features in the evolution of the ribokinase superfamily.     

Crystal structure of the DNA-binding domain of BldD, a central regulator of aerial mycelium formation in Streptomyces coelicolor A3(2)

BldD is a central regulator of the developmental process in Streptomyces coelicolor. The 1.8 angstroms resolution structure of the DNA-binding domain of BldD (BldDN) reveals that BldDN forms a compact globular domain composed of four helices (alpha1-alpha4) containing a helix-turn-helix motif (alpha2-alpha3) resembling that of the DNA-binding domain of lambda repressor. The BldDN/DNA complex model led us to design a series of mutants, which revealed the important role of alpha3 and the 'turn' region between alpha2 and alpha3 for DNA recognition. Based on the fact that BldD occupies two operator sites of bldN and whiG and shows significant disparity in the affinity toward the two operator sites when they are disconnected, we propose a model of cooperative binding, which means that the binding of one BldD dimer to the high affinity site facilitates that of the second BldD dimer to the low affinity site. In addition, structural and mutational investigation reveals that the Tyr62Cys mutation, found in the first-identified bldD mutant, can destabilize BldD structure by disrupting the hydrophobic core.     

Roles of Met-34, Cys-64, and Arg-75 in the assembly of human connexin 26. Implication for key amino acid residues for channel formation and function

Connexins form a family of membrane proteins that assemble into communication channels and directly connect the cytoplasms of adjoining cells. Malfunctioning of connexin channels often cause disease, such as the mutations M34T and R75W in human connexin 26, which are associated with hereditary deafness. Another residue known to be essential for normal channel activity in the connexin is Cys-64. To obtain structural and functional insights of connexin 26, we studied the roles of these three residues by expressing mutant connexins in insect Sf9 and HeLa cells. The M34T and M34A mutants both formed gap junction plaques, but dye transfer assays showed that the M34A mutant had a significantly reduced permeability, suggesting that for proper channel function a side chain of adequate size is required at this position. We propose that Met-34 is located in the innermost helix of the channel, where it ensures a fully open channel structure via interactions with other transmembrane helices. Gap junction channels formed by the R75W and R75D mutants dissociated upon solubilization in dodecyl maltoside, whereas the R75A mutant remained hexameric. All gap junctions formed by Arg-75 mutants also showed only negligible activity in dye transfer experiments. These results suggest that residue Arg-75 plays a role in subunit interactions needed to retain a functional and stable connexin hexamer. The C64S mutant was suggested to be defective in oligomerization and/or protein folding even in the presence of wild-type connexin.     

Allosteric free energy changes at the alpha 1 beta 2 interface of human hemoglobin probed by proton exchange of Trp beta 37

The energetic changes that occur on ligand binding in human hemoglobin have been investigated by measurements of the exchange rates of the indole proton of Trpbeta37(C3). The Trpbeta37 residues are located in helices C of the beta-subunits and are involved in contacts with the segments FG of the alpha-subunits at the interdimeric alpha1beta2 and alpha2beta1 interfaces of the hemoglobin tetramer. In the quaternary structure change that accompanies ligand binding to hemoglobin, these contacts undergo minimal changes in relative orientation and in packing, thereby acting as hinges, or flexible joints. The exchange rates of the indole proton of Trpbeta37(C3) were measured by nuclear magnetic resonance spectroscopy, in both deoxygenated and ligated hemoglobin. The results indicate that, at 15 degrees C, the exchange rate is increased from 9.0. 10(-6) to 3.3. 10(-4) s(-1) upon ligand binding to hemoglobin. This change suggests that the structural units at the hinge regions of the alpha1beta2/alpha2beta1 interfaces containing Trpbeta37(C3) are specifically stabilized in unligated hemoglobin, and experience a change in structural free energy of approximately 4 kcal/(mol tetramer) upon ligand binding. Therefore, the hinge regions of the alpha1beta2/alpha2beta1 interfaces could play a role in the transmission of free energy through the hemoglobin molecule during its allosteric transition.     

The molecular evolution of bacterial alkaline phosphatase: correlating variation among enteric bacteria to experimental manipulations of the protein

The phylogenetic distribution of the gene coding for bacterial alkaline phosphatase (phoA) was examined in nine species of enteric bacteria closely related to Escherichia coli. The nucleotide and protein sequences from the E. fergusonii and Serratia marcescens genes are presented. The spatial distribution of replaced amino acid residues in the aligned sequences is shown to be highly nonrandom and can be correlated with specific regions within the tertiary structure of the protein. There is an avoidance of replacements within the beta sheet of the protein, and there is an excess of replacements elsewhere, particularly in solvent-exposed residues. In addition, all positions across alpha helices do not accept replacements with equal frequency; there is a bias toward acceptance of replacements in the carboxyl ends of helices. To examine this further, mutations within the E. coli phoA gene were created using site-directed mutagenesis. The patterns seen from the sequence comparisons were verified in the laboratory-created mutants. The average activity of mutations within or near the beta sheet was approximately one-third of that within or near alpha helices, and multiple mutations within the carboxyl ends of alpha helices always possessed greater activity than did multiple mutations within the corresponding amino ends. The results indicate that identifiable regions within the protein are under different selective pressures and are therefore evolving at different rates.     

Insights into the oncogenic effects of /PIK3CA/ mutations from the structure of p110α/p85α

Phosphatidylinositide-3-kinases (PI3K) initiate a number of signaling pathways by recruiting other kinases, such as Akt, to the plasma membrane. One of the isoforms, PI3Kα, is an oncogene frequently mutated in several cancer types. These mutations increase PI3K kinase activity, leading to increased cell survival, cell motility, cell metabolism, and cell cycle progression. The structure of the complex between the catalytic subunit of PI3Kα, p110α, and a portion of its regulatory subunit, p85α reveals that the majority of the oncogenic mutations occur at the interfaces between p110 domains and between p110 and p85 domains. At these positions, mutations disrupt interactions resulting in changes in the kinase domain that may increase enzymatic activity. The structure also suggests that interaction with the membrane is mediated by one of the p85 domains (iSH2). These findings may provide novel structural loci for the design of new anti-cancer drugs.     

Diverse gap junctions modulate distinct mechanisms for fiber cell formation during lens development and cataractogenesis

Different mutations of alpha3 connexin (Cx46 or Gja8) and alpha8 connexin (Cx50 or Gja8), subunits of lens gap junction channels, cause a variety of cataracts via unknown mechanisms. We identified a dominant cataractous mouse line (L1), caused by a missense alpha8 connexin mutation that resulted in the expression of alpha8-S50P mutant proteins. Histology studies showed that primary lens fiber cells failed to fully elongate in heterozygous alpha8(S50P/+) embryonic lenses, but not in homozygous alpha8(S50P/S50P), alpha8-/- and alpha3-/- alpha8-/- mutant embryonic lenses. We hypothesized that alpha8-S50P mutant subunits interacted with wild-type alpha3 or alpha8, or with both subunits to affect fiber cell formation. We found that the combination of mutant alpha8-S50P and wild-type alpha8 subunits specifically inhibited the elongation of primary fiber cells, while the combination of alpha8-S50P and wild-type alpha3 subunits disrupted the formation of secondary fiber cells. Thus, this work provides the first in vivo evidence that distinct mechanisms, modulated by diverse gap junctions, control the formation of primary and secondary fiber cells during lens development. This explains why and how different connexin mutations lead to a variety of cataracts. The principle of this explanation can also be applied to mutations of other connexin isoforms that cause different diseases in other organs.     

Structural and sequence characteristics of long alpha helices in globular proteins.

Elucidation of the detailed structural features and sequence requirements for alpha helices of various lengths could be very important in understanding secondary structure formation in proteins and, hence, in the protein folding mechanism. An algorithm to characterize the geometry of an alpha helix from its C(alpha) coordinates has been developed and used to analyze the structures of long alpha helices (number of residues > or = 25) found in globular proteins, the crystal structure coordinates of which are available from the Brookhaven Protein Data Bank. All long alpha helices can be unambiguously characterized as belonging to one of three classes: linear, curved, or kinked, with a majority being curved. Analysis of the sequences of these helices reveals that the long alpha helices have unique sequence characteristics that distinguish them from the short alpha helices in globular proteins. The distribution and statistical propensities of individual amino acids to occur in long alpha helices are different from those found in short alpha helices, with amino acids having longer side chains and/or having a greater number of functional groups occurring more frequently in these helices. The sequences of the long alpha helices can be correlated with their gross structural features, i.e., whether they are curved, linear, or kinked, and in case of the curved helices, with their curvature.     

The structural context of disease-causing mutations in gap junctions

Gap junctions form intercellular channels that mediate metabolic and electrical signaling between neighboring cells in a tissue. Lack of an atomic resolution structure of the gap junction has made it difficult to identify interactions that stabilize its transmembrane domain. Using a recently computed model of this domain, which specifies the locations of each amino acid, we postulated the existence of several interactions and tested them experimentally. We introduced mutations within the transmembrane domain of the gap junction-forming protein connexin that were previously implicated in genetic diseases and that apparently destabilized the gap junction, as evidenced here by the absence of the protein from the sites of cell-cell apposition. The model structure helped identify positions on adjacent helices where second-site mutations restored membrane localization, revealing possible interactions between residue pairs. We thus identified two putative salt bridges and one pair involved in packing interactions in which one disease-causing mutation suppressed the effects of another. These results seem to reveal some of the physical forces that underlie the structural stability of the gap junction transmembrane domain and suggest that abrogation of such interactions bring about some of the effects of disease-causing mutations.     

A structural mechanism of integrin alpha(IIb)beta(3) "inside-out" activation as regulated by its cytoplasmic face

Activation of the ligand binding function of integrin heterodimers requires transmission of an "inside-out" signal from their small intracellular segments to their large extracellular domains. The structure of the cytoplasmic domain of a prototypic integrin alpha(IIb)beta(3) has been solved by NMR and reveals multiple hydrophobic and electrostatic contacts within the membrane-proximal helices of its alpha and the beta cytoplasmic tails. The interface interactions are disrupted by point mutations or the cytoskeletal protein talin that are known to activate the receptor. These results provide a structural mechanism by which a handshake between the alpha and the beta cytoplasmic tails restrains the integrin in a resting state and unclasping of this interaction triggers the inside-out conformational signal that leads to receptor activation.     

Analysis of Mutations in the Pore-Forming Region Essential for Insecticidal Activity of a Bacillus thuringiensis δ-Endotoxin

The Bacillus thuringiensis insecticidal delta-endotoxins have a three-domain structure, with the seven amphipathic helices which comprise domain I being essential for toxicity. To better define the function of these helices in membrane insertion and toxicity, either site-directed or random mutagenesis of two regions was performed. Thirty-nucleotide segments in the B. thuringiensis cry1Ac1 gene, encoding parts of helix alpha4 and the loop connecting helices alpha4 and alpha5, were randomly mutagenized. This hydrophobic region of the toxin probably inserts into the membrane as a hairpin. Site-directed mutations were also created in specific surface residues of helix alpha3 in order to increase its hydrophobicity. Among 12 random mutations in helix alpha4, 5 resulted in the total loss of toxicity for Manduca sexta and Heliothis virescens, another caused a significant increase in toxicity, and one resulted in decreased toxicity. None of the nontoxic mutants was altered in toxin stability, binding of toxin to a membrane protein, or the ability of the toxin to aggregate in the membrane. Mutations in the loop connecting helices alpha4 and alpha5 did not affect toxicity, nor did mutations in alpha3, which should have enhanced the hydrophobic properties of this helix. In contrast to mutations in helix alpha5, those in helix alpha4 which inactivated the toxin did not affect its capacity to oligomerize in the membrane. Despite the formation of oligomers, there was no ion flow as measured by light scattering. Helix alpha5 is important for oligomerization and perhaps has other functions, whereas helix alpha4 must have a more direct role in establishing the properties of the channel.     

Geometric Similarities of Protein-Protein Interfaces at Atomic Resolution Are Only Observed within Homologous Families: An Exhaustive Structural Classification Study

To elucidate the structural basis of the diversity and universality in protein-protein interactions, an exhaustive all-against-all structural comparison of all known protein interfaces in the Protein Data Bank was performed at atomic resolution. After similar interfaces were clustered, approximately 20,000 structural motifs with at least two members were identified, out of which 3678 motifs consisted of at least 10 interfaces. Except for some trivial interfaces involving single α helices, almost all motifs were found to be confined within single protein families. Furthermore, the interaction partners of each motif were found to be very limited, and, accordingly, the interaction networks of the motifs tend to be small and are much more restricted than the binding sites for small ligand molecules. These findings suggest that, at the level of atomic structures, protein-protein interactions are precisely designed; hence, protein interfaces with multiple interacting partners should involve incompletely overlapping multiple interfaces and/or accommodate structural changes upon binding to their targets.     

Crystal structure of the S.cerevisiae exocyst component Exo70p

The exocyst is an evolutionarily conserved multiprotein complex required for the targeting and docking of post-Golgi vesicles to the plasma membrane. Through its interactions with a variety of proteins, including small GTPases, the exocyst is thought to integrate signals from the cell and signal that vesicles arriving at the plasma membrane are ready for fusion. Here we describe the three-dimensional crystal structure of one of the components of the exocyst, Exo70p, from Saccharomyces cerevisiae at 3.5A resolution. Exo70p binds the small GTPase Rho3p in a GTP-dependent manner with an equilibrium dissociation constant of approximately 70 microM. Exo70p is an extended rod approximately 155 angstroms in length composed principally of alpha helices, and is a novel fold. The structure provides a first view of the Exo70 protein family and provides a framework to study the molecular function of this exocyst component.     

Interfacial and distal-heme pocket mutations exhibit additive effects on the structure and function of hemoglobin

Protein engineering strategies seek to develop a hemoglobin-based oxygen carrier with optimized functional properties, including (i) an appropriate O 2 affinity, (ii) high cooperativity, (iii) limited NO reactivity, and (iv) a diminished rate of auto-oxidation. The mutations alphaL29F, alphaL29W, alphaV96W and betaN108K individually impart some of these traits and in combinations produce hemoglobin molecules with interesting ligand-binding and allosteric properties. Studies of the ligand-binding properties and solution structures of single and multiple mutants have been performed. The aromatic side chains placed in the distal-heme pocket environment affect the intrinsic ligand-binding properties of the mutated subunit itself, beyond what can be explained by allostery, and these changes are accompanied by local structural perturbations. In contrast, hemoglobins with mutations in the alpha 1beta 1 and alpha 1beta 2 interfaces display functional properties of both "R"- and "T"-state tetramers because the equilibrium between quaternary states is altered. These mutations are accompanied by global structural perturbations, suggesting an indirect, allostery-driven cause for their effects. Combinations of the distal-heme pocket and interfacial mutations exhibit additive effects in both structural and functional properties, contribute to our understanding of allostery, and advance protein-engineering methods for manipulating the O 2 binding properties of the hemoglobin molecule.