Domain shuffling of a highly mutable ligand-binding fold drives adhesin generation across the bacterial kingdom.

Bacterial fibrillar adhesins are specialized extracellular polypeptides that promote the attachment of bacteria to the surfaces of other cells or materials. Adhesin-mediated interactions are critical for the establishment and persistence of stable bacterial populations within diverse environmental niches and are important determinants of virulence. The fibronectin (Fn)-binding fibrillar adhesin CshA, and its paralogue CshB, play important roles in host colonization by the oral commensal and opportunistic pathogen Streptococcus gordonii. As paralogues are often catalysts for functional diversification, we have probed the early stages of structural and functional divergence in Csh proteins by determining the X-ray crystal structure of the CshB adhesive domain NR2 and characterizing its Fn-binding properties in vitro. Despite sharing a common fold, CshB_NR2 displays an ~1.7-fold reduction in Fn-binding affinity relative to CshA_NR2. This correlates with reduced electrostatic charge in the Fn-binding cleft. Complementary bioinformatic studies reveal that homologues of CshA/B_NR2 domains are widely distributed in both Gram-positive and Gram-negative bacteria, where they are found housed within functionally cryptic multi-domain polypeptides. Our findings are consistent with the classification of Csh adhesins and their relatives as members of the recently defined polymer adhesin domain (PAD) family of bacterial proteins.

Activation by substoichiometric inhibition.

Startling reports described the paradoxical triggering of the human mitogen-activated protein kinase pathway when a small-molecule inhibitor specifically inactivates the BRAF V600E protein kinase but not wt-BRAF. We performed a conceptual analysis of the general phenomenon "activation by inhibition" using bacterial and human HtrA proteases as models. Our data suggest a clear explanation that is based on the classic biochemical principles of allostery and cooperativity. Although substoichiometric occupancy of inhibitor binding sites results in partial inhibition, this effect is overrun by a concomitant activation of unliganded binding sites. Therefore, when an inhibitor of a cooperative enzyme does not reach saturating levels, a common scenario during drug administration, it may cause the contrary of the desired effect. The implications for drug development are discussed.

The streptococcal multidomain fibrillar adhesin CshA has an elongated polymeric architecture.

The cell surfaces of many bacteria carry filamentous polypeptides termed adhesins that enable binding to both biotic and abiotic surfaces. Surface adherence is facilitated by the exquisite selectivity of the adhesins for their cognate ligands or receptors and is a key step in niche or host colonization and pathogenicity. Streptococcus gordonii is a primary colonizer of the human oral cavity and an opportunistic pathogen, as well as a leading cause of infective endocarditis in humans. The fibrillar adhesin CshA is an important determinant of S. gordonii adherence, forming peritrichous fibrils on its surface that bind host cells and other microorganisms. CshA possesses a distinctive multidomain architecture comprising an N-terminal target-binding region fused to 17 repeat domains (RDs) that are each ∼100 amino acids long. Here, using structural and biophysical methods, we demonstrate that the intact CshA repeat region (CshA_RD1-17, domains 1-17) forms an extended polymeric monomer in solution. We recombinantly produced a subset of CshA RDs and found that they differ in stability and unfolding behavior. The NMR structure of CshA_RD13 revealed a hitherto unreported all ß-fold, flanked by disordered interdomain linkers. These findings, in tandem with complementary hydrodynamic studies of CshA_RD1-17, indicate that this polypeptide possesses a highly unusual dynamic transitory structure characterized by alternating regions of order and disorder. This architecture provides flexibility for the adhesive tip of the CshA fibril to maintain bacterial attachment that withstands shear forces within the human host. It may also help mitigate deleterious folding events between neighboring RDs that share significant structural identity without compromising mechanical stability.

Toward a detailed description of the pathways of allosteric communication in the GroEL chaperonin through atomistic simulation.

GroEL, along with its coprotein GroES, is essential for ensuring the correct folding of unfolded or newly synthesized proteins in bacteria. GroEL is a complex, allosteric molecule, composed of two heptameric rings stacked back to back, that undergoes large structural changes during its reaction cycle. These structural changes are driven by the cooperative binding and subsequent hydrolysis of ATP, by GroEL. Despite numerous previous studies, the precise mechanisms of allosteric communication and the associated structural changes remain elusive. In this paper, we describe a series of all-atom, unbiased, molecular dynamics simulations over relatively long (50-100 ns) time scales of a single, isolated GroEL subunit and also a heptameric GroEL ring, in the presence and absence of ATP. Combined with results from a distance restraint-biased simulation of the single ring, the atomistic details of the earliest stages of ATP-driven structural changes within this complex molecule are illuminated. Our results are in broad agreement with previous modeling studies of isolated subunits and with a coarse-grained, forcing simulation of the single ring. These are the first reported all-atom simulations of the GroEL single-ring complex and provide a unique insight into the role of charged residues K80, K277, R284, R285, and E388 at the subunit interface in transmission of the allosteric signal. These simulations also demonstrate the feasibility of performing all-atom simulations of very large systems on sufficiently long time scales on typical high performance computing facilities to show the origins of the earliest events in biologically relevant processes.

Mapping the road to recovery: the ClpB/Hsp104 molecular chaperone.

The AAA(+)-ATPases are a family of molecular motors which have been seconded into a plethora of cellular tasks. One subset, the Hsp100 molecular chaperones, are general protein remodellers that help to maintain the integrity of the cellular proteome by means of protein destruction or resurrection. In this review we focus on one family of Hsp100s, the homologous ClpB and Hsp104 molecular chaperones that convey thermotolerance by resolubilising and rescuing proteins from aggregates. We explore how the nucleotide binding and hydrolysis properties at the twelve nucleotide-binding domains of these hexameric rings are coupled to protein disaggregation, highlighting similarities and differences between ClpB and Hsp104.

An ACP structural switch: conformational differences between the apo and holo forms of the actinorhodin polyketide synthase acyl carrier protein.

The actinorhodin (act) synthase acyl carrier protein (ACP) from Streptomyces coelicolor plays a central role in polyketide biosynthesis. Polyketide intermediates are bound to the free sulfhydryl group of a phosphopantetheine arm that is covalently linked to a conserved serine residue in the holo form of the ACP. The solution NMR structures of both the apo and holo forms of the ACP are reported, which represents the first high resolution comparison of these two forms of an ACP. Ensembles of twenty apo and holo structures were calculated and yielded atomic root mean square deviations of well-ordered backbone atoms to the average coordinates of 0.37 and 0.42 A, respectively. Three restraints defining the protein to the phosphopantetheine interface were identified. Comparison of the apo and holo forms revealed previously undetected conformational changes. Helix III moved towards helix II (contraction of the ACP), and Leu43 on helix II subtly switched from being solvent exposed to forming intramolecular interactions with the newly added phosphopantetheine side chain. Tryptophan fluorescence and S. coelicolor fatty acid synthase (FAS) holo-synthase (ACPS) assays indicated that apo-ACP has a twofold higher affinity (K(d) of 1.1 muM) than holo-ACP (K(d) of 2.1 muM) for ACPS. Site-directed mutagenesis of Leu43 and Asp62 revealed that both mutations affect binding, but have differential affects on modification by ACPS. Leu43 mutations in particular strongly modulate binding affinity for ACPS. Comparison of apo- and holo-ACP structures with known models of the Bacillus subtilis FAS ACP-holo-acyl carrier protein synthase (ACPS) complex suggests that conformational modulation of helix II and III between apo- and holo-ACP could play a role in dissociation of the ACP-ACPS complex.

A kinetic analysis of the nucleotide-induced allosteric transitions in a single-ring mutant of GroEL.

The function of GroE requires a complex system of allosteric communication driven by protein-nucleotide interactions. These rearrangements couple the binding and hydrolysis of ATP to an overall reaction cycle in which substrate proteins are bound, encapsulated and released. Positive cooperativity with respect to ATP binding occurs within one heptameric ring of GroEL, while negative cooperativity between the two rings generates an inherent asymmetry between the two rings. A previously engineered mutant of GroEL in which the ring-ring contacts are broken gives rise to a single-ring version of the wild-type chaperonin (SR1). We have studied the kinetics of the nucleotide-induced conformational changes in a single-tryptophan variant of SR1 (Y485W-SR1) and compared the resulting data with those we reported previously for the double-ring, single-tryptophan variant of wild-type GroEL (Y485W-GroEL). Remarkably, the parting of the rings does not have a major effect on the conformational changes occurring within the heptameric ring and a kinetic model is presented to describe the sequence of structural rearrangements that occur upon ATP binding to the SR1 molecule. The observation that both the ATP-induced and ADP-induced conformational rearrangements occur more rapidly in the SR1 than they do in wild-type GroEL, indicates that intra-ring conformational changes in the double-ring structure must overcome conformational constraints provided by the presence of the second ring. Lastly, the data presented here imply a role for inter-ring allostery in controlling the dissociation-association behaviour of the GroES co-protein in the overall reaction cycle.

Characterisation of a GroEL single-ring mutant that supports growth of Escherichia coli and has GroES-dependent ATPase activity.

Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.

Structure and malonyl CoA-ACP transacylase binding of streptomyces coelicolor fatty acid synthase acyl carrier protein.

Malonylation of an acyl carrier protein (ACP) by malonyl Coenzyme A-ACP transacylase (MCAT) is fundamental to bacterial fatty acid biosynthesis. Here, we report the structure of the Steptomyces coelicolor (Sc) fatty acid synthase (FAS) ACP and studies of its binding to MCAT. The carrier protein adopts an alpha-helical bundle structure common to other known carrier proteins. The Sc FAS ACP shows close structural homology with other fatty acid ACPs and less similarity with Sc actinorhodin (act) polyketide synthase (PKS) ACP where the orientation of helix I differs. NMR experiments were used to map the binding of ACP to MCAT. This data suggests that Sc FAS ACP interacts with MCAT through the negatively charged helix II of ACP, consistent with proposed models for ACP recognition by other FAS enzymes. Differential roles for residues at the interface are demonstrated using site-directed mutagenesis and in vitro assays. MCAT has been suggested, moreover, to participate in bacterial polyketide synthesis in vivo. We demonstrate that the affinity of the polyketide synthase ACP for MCAT is lower than that of the FAS ACP. Mutagenesis of homologous helix II residues on the polyketide synthase ACP suggests that the PKS ACP may bind to MCAT in a different manner than the FAS counterpart.

Biochemical and functional analysis of the assembly of full-length Sup35p and its prion-forming domain.

The protein Sup35 has prion properties. Its aggregation is at the origin of the [PSI(+)] trait in Saccharomyces cerevisiae. In vitro, the N-terminal domain of Sup35p alone or with the middle domain assembles into fibrils that exhibit the characteristics of amyloids. The vast majority of in vitro studies on the assembly of Sup35p have been performed using Sup35pNM, as fibrils made of Sup35pNM assembled in vitro propagate [PSI(+)] when reintroduced into yeast cells. Little is known about the assembly of full-length Sup35p and the role of the functional C-terminal domain of the protein. Here we report a systematic comparison of the biochemical and assembly properties of full-length Sup35p and Sup35pNM. We show that the native structure of the C-terminal domain is retained within the fibrils. We determined the size of Sup35p nuclei and the critical concentration for assembly that both differ from that of Sup35pNM. We demonstrate that Sup35pNM co-assembles with the full-length protein and that fibrils made of Sup35p or Sup35pNM seed the assembly of soluble Sup35pNM and Sup35p with different efficiencies. Finally, we show that fibrils made of full-length Sup35p induce with higher efficiency [PSI(+)] appearance as compared with those made of Sup35pNM. Our findings reveal differences and similarities in the assembly of Sup35p and its NM fragment and validate the use of Sup35pNM in studying some aspects of Sup35p aggregation but also underline the importance of using full-length Sup35p in studying prion propagation both in vivo and in vitro.

Elucidation of steps in the capture of a protein substrate for efficient encapsulation by GroE.

We have identified five structural rearrangements in GroEL induced by the ordered binding of ATP and GroES. The first discernable rearrangement (designated T --> R(1)) is a rapid, cooperative transition that appears not to be functionally communicated to the apical domain. In the second (R(1) --> R(2)) step, a state is formed that binds GroES weakly in a rapid, diffusion-limited process. However, a second optical signal, carried by a protein substrate bound to GroEL, responds neither to formation of the R(2) state nor to the binding of GroES. This result strongly implies that the substrate protein remains bound to the inner walls of the initially formed GroEL.GroES cavity, and is not yet displaced from its sites of interaction with GroEL. In the next rearrangement (R(2).GroES --> R(3).GroES) the strength of interaction between GroEL and GroES is greatly enhanced, and there is a large and coincident loss of fluorescence-signal intensity in the labeled protein substrate, indicating that there is either a displacement from its binding sites on GroEL or at least a significant change of environment. These results are consistent with a mechanism in which the shift in orientation of GroEL apical domains between that seen in the apo-protein and stable GroEL.GroES complexes is highly ordered, and transient conformational intermediates permit the association of GroES before the displacement of bound polypeptide. This ensures efficient encapsulation of the polypeptide within the GroEL central cavity underneath GroES.

Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein.

During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and 1H NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.

Identification of a major inter-ring coupling step in the GroEL reaction cycle.

It has been shown previously that the double-ring structure of GroEL can be converted to a single-ring species by site-directed amino acid replacements at the ring interface and that the resultant molecule retains many of the crucial chaperonin properties; it is structurally stable, hydrolytically active, and can bind both the co-chaperonin, GroES, and unfolded substrate proteins. By comparing the behavior of the double- and single-ring structures in response to nucleotide binding and hydrolysis, we elucidate steps in the ATP-driven reaction cycle at which there is conformational coupling between the rings. Remarkably, the parting of the rings has little effect either on the thermodynamic properties of ATP binding or on the ATP-induced conformational changes prior to hydrolysis. However, there is a marked effect on the rate-limiting process in the steady-state cycle; a step that is coincident with bond cleavage in ATP. The effect of the ring-ring interaction is to increase its activation enthalpy from 42.0 to 94.2 kJ/mol. These results show that the major conformational coupling step, where structural rearrangements in one ring are propagated to the other, is the slowest process the ATPase cycle of GroEL.