Comprehensive Analysis of HAMP Domains: Implications for Transmembrane Signal Transduction

Homodimeric receptors with one or two transmembrane (TM) segments per monomer are universal to life and represent the largest and most diverse group of cellular TM receptors. They frequently share domain types across phyla and, in some cases, have been recombined experimentally into functional chimeras (e.g., the bacterial aspartate chemoreceptor with the human insulin receptor), suggesting that they have a common mechanism. The nature of this mechanism, however, is still being debated. We have proposed a new model for transduction mechanism by axial helix rotation, based on the structure of a widespread domain, HAMP, that frequently occurs in direct continuation of the last TM segment, primarily in histidine kinases and chemoreceptors. Here we show by statistical analysis that HAMP domain sequences have biophysical properties compatible with the two conformations proposed by the model. The analysis also identifies three networks of coevolving residues, which allow the mechanism to subdivide into individual steps. The most extended of these networks is specific for membrane-bound HAMP domains and most likely accepts the signal from the TM helices. In a classification based on sequence clustering, these HAMPs form a central supercluster, surrounded by smaller clusters of divergent HAMPs, which typically combine into arrays of up to 31 consecutive copies and accept conformational input from other HAMP domains. Unexpectedly, the classification shows a division between domains of histidine kinases and those of chemoreceptors; thus, except for a few versatile lineages, HAMP domains are largely specific for one particular output domain. Within proteins using a given output domain, HAMP domains also show extensive coevolution with histidine kinases, but not with chemoreceptors. We attribute the greater capability for recombination among chemoreceptors to their acquisition of a reversible modification system, which acts as a capacitor for the initially deleterious effects of combining domains optimized in different contexts.     

A galaxy of folds

Many protein classification systems capture homologous relationships by grouping domains into families and superfamilies on the basis of sequence similarity. Superfamilies with similar 3D structures are further grouped into folds. In the absence of discernable sequence similarity, these structural similarities were long thought to have originated independently, by convergent evolution. However, the growth of databases and advances in sequence comparison methods have led to the discovery of many distant evolutionary relationships that transcend the boundaries of superfamilies and folds. To investigate the contributions of convergent versus divergent evolution in the origin of protein folds, we clustered representative domains of known structure by their sequence similarity, treating them as point masses in a virtual 2D space which attract or repel each other depending on their pairwise sequence similarities. As expected, families in the same superfamily form tight clusters. But often, superfamilies of the same fold are linked with each other, suggesting that the entire fold evolved from an ancient prototype. Strikingly, some links connect superfamilies with different folds. They arise from modular peptide fragments of between 20 and 40 residues that co‐occur in the connected folds in disparate structural contexts. These may be descendants of an ancestral pool of peptide modules that evolved as cofactors in the RNA world and from which the first folded proteins arose by amplification and recombination. Our galaxy of folds summarizes, in a single image, most known and many yet undescribed homologous relationships between protein superfamilies, providing new insights into the evolution of protein domains.     

Model structure of the prototypical non-fimbrial adhesin YadA of Yersinia enterocolitica

Non-fimbrial adhesins, such as Yersinia YadA, Moraxella UspA1 and A2, Haemophilus Hia and Hsf, or Bartonella BadA represent an important class of molecules by which pathogenic proteobacteria adhere to their hosts. They form trimeric surface structures with a head-stalk-anchor architecture. Whereas head and stalk domains are diverse and appear (frequently repetitively) in different combinations, the anchor domains are homologous and display the properties of autotransporters. We have built a molecular model for the prototypical non-fimbrial adhesin, YadA, by combining the crystal structure of the head (PDB:1P9H) with theoretical models for the stalk and the anchor. The head domain is a single-stranded, left-handed beta-helix, connected to the stalk by a conserved trimerization element (the neck). The stalk consists of a right-handed coiled coil, containing ten 15-residue repeats with a C-terminal stutter (insertion of four residues). The stalk continues into the conserved anchor domain, which is formed by four heptads of a left-handed coiled coil, followed by four transmembrane beta-strands. Our model of the YadA coiled coil, generated with the program BeammotifCC, combines these periodicities into a structure that starts with a pronounced right-handed supercoil and ends with a canonical, left-handed conformation. The last two heptads of the coiled coil are located within a 12-stranded beta-barrel, formed by trimerization of the four transmembrane beta-strands in each monomer. We propose that this pore assembles in the outer membrane to form the opening through which the monomer chains exit the cell. After export is completed, the fiber folds and the pore is occluded by the coiled coil. Our model explains how these proteins can act as autotransporters in the absence of any homology to classical, single-chain autotransporters.     

AAA proteins

AAA proteins have entered the molecular realm after being known primarily for their wide range of different functions. Structural studies have highlighted the organization of their constituent ATPase domains and indicate that hexamerization in combination with unfoldase activity is a common underlying feature of this ubiquitous protein family.     

Phylogenetic analysis of AAA proteins

AAA ATPases form a large protein family with manifold cellular roles. They belong to the AAA+ superfamily of ringshaped P-loop NTPases, which exert their activity through the energy-dependent unfolding of macromolecules. Phylogenetic analyses have suggested the existence of five major clades of AAA domains (proteasome subunits, metalloproteases, domains D1 and D2 of ATPases with two AAA domains, and the MSP1/katanin/spastin group), as well as a number of deeply branching minor clades. These analyses however have been characterized by a lack of consistency in defining the boundaries of the AAA family. We have used cluster analysis to delineate unambiguously the group of AAA sequences within the AAA+ superfamily. Phylogenetic and cluster analysis of this sequence set revealed the existence of a sixth major AAA clade, comprising the mitochondrial, membrane-bound protein BCS1 and its homologues. In addition, we identified several deep branches consisting mainly of hypothetical proteins resulting from genomic projects. Analysis of the AAA N-domains provided direct support for the obtained phylogeny for most branches, but revealed some deep splits that had not been apparent from phylogenetic analysis and some unexpected similarities between distant clades. It also revealed highly degenerate D1 domains in plant MSP1 sequences and in at least one deeply branching group of hypothetical proteins (YC46), showing that AAA proteins with two ATPase domains arose at least three times independently.     

Thermoplasma acidophilum TAA43 is an archaeal member of the eukaryotic meiotic branch of AAA ATPases

Sequencing of the Thermoplasma acidophilum genome revealed a new gene, taa43 , which codes for a 43-kDa protein containing one AAA domain; we therefore termed it Thermoplasma AAA ATPase of 43 kDa (TAA43). Close homologs of TAA43 are found only in related Thermoplasmales, e.g. T. volcanium and Ferroplasma acidarmanus , but not in other Archaea. A detailed phylogenetic analysis showed that TAA43 and its homologs belong to the 'meiotic' branch of the AAA family. Although AAA proteins usually assemble into high-molecular-weight complexes, native TAA43 is predominantly dimeric except for a minor fraction eluting in the void volume of a sizing column. Wild-type and mutant TAA43 proteins were overexpressed in Escherichia coli , purified as dimers and characterized functionally. Since the canonical proteasome activating nucleotidase is not present in Thermoplasmales, TAA43 was tested for stimulation of proteasome activity, which was, however, not detected. Interestingly, immunoprecipitation analysis with TAA43 specific antibodies found a fraction of native TAA43 associated with Thermoplasma ribosomal proteins.     

The structure of E. coli IgG-binding protein D suggests a general model for bending and binding in trimeric autotransporter adhesins

The Escherichia coli Ig-binding (Eib) proteins are trimeric autotransporter adhesins (TAAs) and receptors for IgG Fc. We present the structure of a large fragment of the passenger domain of EibD, the first TAA structure to have both a YadA-like head domain and the entire coiled-coil stalk. The stalk begins as a right-handed superhelix, but switches handedness halfway down. An unexpected β-minidomain joins the two and inserts a ～120° rotation such that there is no net twist between the beginning and end of the stalk. This may be important in folding and autotransport. The surprisingly large cavities we found in EibD and other TAAs may explain how TAAs bend to bind their ligands. We identified how IgA and IgG bind and modeled the EibD-IgG Fc complex. We further show that EibD promotes autoagglutination and biofilm formation and forms a fibrillar layer covering the cell surface making zipper-like contacts between cells.     

Gene duplication of the eight-stranded beta-barrel OmpX produces a functional pore: a scenario for the evolution of transmembrane beta-barrels

The repeating unit of outer membrane beta-barrels from Gram-negative bacteria is the beta-hairpin, and representatives of this protein family always have an even strand number between eight and 22. Two dominant structural forms have eight and 16 strands, respectively, suggesting gene duplication as a possible mechanism for their evolution. We duplicated the sequence of OmpX, an eight-stranded beta-barrel protein of known structure, and obtained a beta-barrel, designated Omp2X, which can fold in vitro and in vivo. Using single-channel conductance measurements and PEG exclusion assays, we found that Omp2X has a pore size similar to that of OmpC, a natural 16-stranded barrel. Fusions of the homologous proteins OmpX, OmpA and OmpW were able to fold in vitro in all combinations tested, revealing that the general propensity to form a beta-barrel is sufficient to evolve larger barrels by simple genetic events.