Blue flickers of hope: secondary structure, dynamics, and putative dimerization interface of the blue-light receptor YtvA from Bacillus subtilis

Bacillus subtilis is capable of responding to various kinds of extracellular, potentially harmful stimuli via a stress response pathway, which involves a signal transduction and integration hub, the stressosome, and finally leads to activation of σ(B). One of the different signals initiating the underlying phosphorylation cascade is blue light. While it is known that the bacterial photoreceptor YtvA is responsible for blue light detection, the intramolecular activation mechanism and the structure of this multidomain protein are unknown. Using solution NMR spectroscopy, we have obtained a near complete backbone assignment of the full-length protein. More importantly, we report relaxation data and data on the solvent accessibility of full-length YtvA in the dark state which are interpreted with respect to secondary structure, the mobility, and the quaternary structure of the protein. Finally, we show that YtvA adopts an elongated domain orientation with LOV-LOV and STAS-STAS interactions on either side.     

Blue news: NTP binding properties of the blue-light sensitive YtvA protein from Bacillus subtilis

The blue-light sensitive protein YtvA from Bacillus subtilis is built of a photoactive, flavin-binding LOV (Light, Oxygen and Voltage) domain and a STAS domain with unknown function. Here we show that YtvA binds a fluorescent derivative of guanosine triphosphate (GTPTR) that can be displaced by both GTP or ATP. Unspecific NTP (N=G or A) binding is supported by the molecular model of YtvA-STAS. Blue-light activation of YtvA results in small and dark-reversible spectroscopic changes for GTPTR, suggesting that light-driven conformational changes are transmitted from the LOV core to the GTPTR binding site. These results support the idea that STAS domains may have a general NTP binding role and open a way to investigate the molecular functionality of YtvA-STAS.     

NTP-binding properties of the blue-light receptor YtvA and effects of the E105L mutation

YtvA is a blue-light-sensing protein from Bacillus subtilis related to plant phototropins. It carries a LOV (light, oxygen and voltage) domain, binding FMN (flavin mononucleotide) as chromophore, and a STAS (sulphate transporters and antisigma-factor antagonists) domain with poorly characterized function. We have recently shown that YtvA binds triphosphate nucleotides (NTP) and highlighted a structural similarity between the STAS domain and small GTP-binding proteins. In this work we further investigated the NTP-binding properties of YtvA, employing a fluorescent derivative of GTP (GTP_TR) and mutagenesis experiments. The main results are as follows: (a) competition experiments indicate that the affinity of YtvA for GTP is much higher than that for GDP and GMP. (b) Blue-light-induced structural changes are transmitted from the LOV core to the NTP-binding cavity, establishing a possible intraprotein signal-transduction pathway. (c) A mutation in the central β-scaffold of the LOV core, E105L, impairs the light-driven spectroscopic changes of bound GTP_TR. This result is supported by circular dichroism data, in that YtvA-E105L does not show the light-induced conformational change in the turn fraction that characterizes YtvA, implying that E105 is functionally important. (d) In the structural model of the LOV-STAS complex, based on docking algorithms, the interface includes the Iβ–Hβ loop on the LOV core, as well as parts of the central β-scaffold. E105 is predicted to interact with the LOV-STAS linker region, suggested to play a role in phototropin signaling. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Structure of the enterococcal T4SS protein PrgL reveals unique dimerization interface in the VirB8 protein family

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Structure-based thermodynamic analysis of caspases reveals key residues for dimerization and activity

Cysteine-dependent aspartic proteases (caspases) are a family of enzymes which play a crucial role in apoptosis. Caspases accumulate in eukaryotic cells in the form of low-activity proenzyme precursors. Proteolytic cleavage of specific sites triggers conformational changes that lead to full activation and thus to the initiation of the apoptotic cascade. Several experimental observations suggest that dimerization is crucial for activity and regulation, but the underlying molecular mechanisms have not yet been completely resolved. In this work, we have used a structure-based thermodynamic analysis method [Edgcomb, S. P., and Murphy, K. P. (2000) Curr. Opin. Biotechnol. 11, 62-66] to calculate the free energy of association and folding for all the caspases and procaspases whose structures are known at present. In all cases, analysis of the single-residue contributions to the dimerization energy shows that 30-50% of the dimer stability originates from the highly specific interaction of 12-14 residues located at the N- and C-termini of the large and small subunits, respectively. Moreover, our calculations indicate that these residues are also critical for stabilizing the conformation of the active site loops, which in turn is crucial for the binding of substrates and inhibitors. Thus, our results help to rationalize the relation between dimerization and activity in this important class of enzymes and can be used as a starting point for an active manipulation of the monomer-dimer equilibrium for preparatory and regulatory purposes.     

Conformational dynamics and the energetics of protein--ligand interactions: role of interdomain loop in human cytochrome P450 reductase

A combination of mutagenesis, calorimetry, kinetics, and small-angle X-ray scattering (SAXS) has been used to study the mechanism of ligand binding energy propagation through human cytochrome P450 reductase (CPR). Remarkably, the energetics of 2',5'-ADP binding to R597 at the FAD-binding domain are affected by mutations taking place at an interdomain loop located 60 A away. Either deletion of a 7 amino acid long segment (T236-G237-E238-E239-S240-S241-I242) or its replacement by poly-proline repeats (5 and 10 residues) results in a significant increase in 2',5'-ADP enthalpy of binding (DeltaHB). This is accompanied by a decrease in the number of thermodynamic microstates available for the ligand-CPR complex. Moreover, the estimated heat capacity change (DeltaCp) for this interaction changes from -220 cal mol-1 K-1 in the wild-type enzyme to -580 cal mol-1 K-1 in the deletion mutant. Pre-steady-state kinetics measurements reveal a 50-fold decrease in the microscopic rate for interdomain (FAD --> FMN) electron transfer in the deletion mutant (kobs = 0.4 s-1). Multiple turnover cytochome c reduction assays indicate that these mutations impair the ability of the FMN-binding domain to shuttle electrons from the FAD-binding domain to the cytochrome partner. Binding of 2',5'-ADP to wild-type CPR triggers a large-scale structural rearrangement resulting in the complex having a more compact domain organization, and the maximum molecular dimension (Dmax) decreases from 110 A in ligand-free enzyme to 100 A in the ligand-bound CPR. The SAXS experiments also demonstrate that what is affected by the mutations is indeed the relative diffusional motion of the domains. Furthemore, ab initio shape reconstruction and homology modeling would suggest that-in the deletion mutant-hindering of domain motion occurs concomitantly with dimerization. The results presented here show that the energetics of this highly localized interaction (2',5'-ADP binding) have a global character, and are highly sensitive to functional structural dynamics involving distal domains. These findings support early theoretical studies which postulate a single protein molecule to be a real, independent thermodynamic ensemble.     

Domain mapping on the human metastasis regulator protein h-Prune reveals a C-terminal dimerization domain

The human orthologue of the Drosophila prune protein (h-Prune) is an interaction partner and regulator of the metastasis suppressor protein NM23-H1 (non-metastatic protein 23). Studies on a cellular breast-cancer model showed that inhibition of the cAMP-specific PDE (phosphodiesterase) activity of h-Prune lowered the incidence of metastasis formation, suggesting that inhibition of h-Prune could be a therapeutic approach towards metastatic tumours. H-Prune shows no sequence similarity with known mammalian PDEs, but instead appears to belong to the DHH (Asp-His-His) superfamily of phosphoesterases. In order to investigate the structure and molecular function of h-Prune, we expressed recombinant h-Prune in a bacterial system. Through sequence analysis and limited proteolysis, we identified domain boundaries and a potential coiled-coil region in a C-terminal cortexillin homology domain. We found that this C-terminal domain mediated h-Prune homodimerization, as well as its interaction with NM23-H1. The PDE catalytic domain of h-Prune was mapped to the N-terminus and shown to be active, even when present in a monomeric form. Our findings indicate that h-Prune is composed of two independent active sites and two interaction sites for the assembly of oligomeric signalling complexes.     

Dynamics of interdomain and intermolecular interactions in mammalian metallothioneins.

The structures of mammalian metallothioneins (MTs), as solved by X-ray crystallography and NMR spectroscopy, all show seven divalent metals bound in two separate domains. The marked differences in metal-mobilities found for the two domains has led to the proposal for a dual role for the two MT metal domains. The tight metal binding in the C-terminal alpha-domain supposedly constitutes the basis for the detoxification of excess heavy metals, while the more labile metals in the N-terminal beta-domain function in the homeostasis of the essential elements zinc and copper. In this overview, we compare the two types of dimers found for MTs and their influence on metal-mobilities. In the presence of excess metal, the N-terminal domain is responsible for the formation of metal-bridged dimers while under aerobic conditions, a specific intermolecular disulfide is formed between the C-terminal domains. Both forms of dimers not only involve different domains for their intermolecular protein interactions, they also exhibit radical differences in the reactive properties of their respective cluster bound metal ions. Since the metal exchange within each domain is also influenced by interdomain interactions, the relative orientation of the domains is also most likely important for MT functions. Thus far, the relative orientation of the two domains could only be obtained from the crystal structure. Here, we present evidence for increased mobility in the linker region as the reason for the lack of interdomain constraints in the solution NMR studies of mammalian MTs.     

Spatial analysis of ectomycorrhizal fungi reveals that root tip communities are structured by competitive interactions

Microbial ecology has made large advances over the last decade, mostly because of improvements in molecular analysis techniques that have enabled the detection and identification of progressively larger numbers of microbial species. However, determining the ecological patterns and processes taking place in communities of microbes remains a significant challenge. Are communities randomly assembled through dispersal and priority effects, or do species interact with each other leading to positive and negative associations? For mycorrhizal fungi, evidence is accumulating that stochastic and competitive interactions between species may both have a role in shaping community structure. Could the methodological approach, which is often incidence based, impact the outcomes detected? Here, we applied an incidence‐based Terminal Restriction Fragment Length Polymorphism (T‐RFLP) database approach to examine species diversity and ecological interactions within a community of ectomycorrhizal (ECM) fungi. Co‐occurrence analysis revealed that the ECM community colonizing root tips was strongly structured by competitive interactions, or ecological processes generating a similar spatial pattern, rather than neutral processes. Analysis of β‐diversity indicated that community structure was significantly more similar (spatially autocorrelated) at distances equal to or <3.41 m. The eight most frequently encountered species in the root tip community of ECM fungi displayed significant competitive interactions with at least one other species, showing that the incidence‐based approach was capable of detecting this sort of ecological information.     

The SufE sulfur-acceptor protein contains a conserved core structure that mediates interdomain interactions in a variety of redox protein complexes

The isc and suf operons in Escherichia coli represent alternative genetic systems optimized to mediate the essential metabolic process of iron-sulfur cluster (Fe-S) assembly under basal or oxidative-stress conditions, respectively. Some of the proteins in these two operons share strong sequence homology, e.g. the cysteine desulfurases IscS and SufS, and presumably play the same role in the oxygen-sensitive assembly process. However, other proteins in these operons share no significant homology and occur in a mutually exclusive manner in Fe-S assembly operons in other organisms (e.g. IscU and SufE). These latter proteins presumably play distinct roles adapted to the different assembly mechanisms used by the two systems. IscU has three invariant cysteine residues that function as a template for Fe-S assembly while accepting a sulfur atom from IscS. SufE, in contrast, does not function as an Fe-S assembly template but has been suggested to function as a shuttle protein that uses a persulfide linkage to a single invariant cysteine residue to transfer a sulfur atom from SufS to an alternative Fe-S assembly template. Here, we present and analyze the 2.0A crystal structure of E.coli SufE. The structure shows that the persulfide-forming cysteine occurs at the tip of a loop with elevated B-factors, where its side-chain is buried from solvent exposure in a hydrophobic cavity located beneath a highly conserved surface. Despite the lack of sequence homology, the core of SufE shows strong structural similarity to IscU, and the sulfur-acceptor site in SufE coincides with the location of the cysteine residues mediating Fe-S cluster assembly in IscU. Thus, a conserved core structure is implicated in mediating the interactions of both SufE and IscU with the mutually homologous cysteine desulfurase enzymes present in their respective operons. A similar core structure is observed in a domain found in a variety of Fe-S cluster containing flavoenzymes including xanthine dehydrogenase, where it also mediates interdomain interactions. Therefore, the core fold of SufE/IscU has been adapted to mediate interdomain interactions in diverse redox protein systems in the course of evolution.     

The molecular basis of cooperativity in protein folding. Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase.

In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.     

Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization.

Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. The structure of a C-terminal truncated form of the human beta subunit has been determined by X-ray crystallography to 1.7 A resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely alpha-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among beta subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation.     

Mechanism and evolution of protein dimerization.

We have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure-kinetics-evolution relationship for a collection of symmetric homodimers classified into three groups: (1) 14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2-state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3-state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain. (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomeric state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers, and can usually predict whether a dimer has domain swapping. The kinetics of dimerization indicates that some dimers which have been assigned in the literature as domain swapping cases, dimerize through the 2-state kinetics, rather than through swapping segments of performed monomers. The compactness profiles indicate a wide spectrum in the kinetics of dimerization: dimers having no intermediate stable monomers; dimers having an intermediate with a stable monomer structure; and dimers having an intermediate with a stable structure in part of the monomer. These correspond to the multiple evolutionary pathways for dimer formation. The evolutionary mechanisms proposed here for dimers are applicable to other oligomers as well.     

Conformational change coupling the dimerization and activation of KSHV protease

The mechanism of herpesviral protease activation upon dimerization was studied using two independent spectroscopic assays augmented by directed mutagenesis. Spectroscopic changes, attributable to dimer interface conformational plasticity, were observed upon dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr). KSHV Pr's dissociation constant of 585 +/- 135 nM at 37 degrees C was measured by a concentration-dependent, 100-fold increase in specific activity to a value of 0.275 +/- 0.023 microM product min(-1) (microM enzyme)(-1). A 4 nm blue-shifted fluorescence emission spectrum and a 25% increase in ellipticity at 222 nm were detected by circular dichroism upon dimer association. This suggested enhanced hydrophobic packing within the dimer interface and/or core, as well as altered secondary structures. To better understand the structure-activity relationship between the monomer and the dimer, KSHV Pr molecules were engineered to remain monomeric via substitution of two separate residues within the dimer interface, L196 and M197. These mutants were proteolytically inactive while exhibiting the spectroscopic signature and thermal stability of wild type, dissociated monomers (T(M) = 75 degrees C). KSHV Pr conformational changes were found to be relevant in vivo, as the autoproteolytic inactivation of KSHV Pr at its dimer disruption site [Pray et al. (1999) J. Mol. Biol. 289, 197-203] was detected in viral particles from KSHV-infected cells. This characterization of structural plasticity suggests that the structure of the KSHV Pr monomer is stable and significantly different from its structure in the dimer. This structural uniqueness should be considered in the development of compounds targeting the dimer interface of KSHV Pr monomers.     

Ligand-mediated dimerization of a carbohydrate-binding molecule reveals a novel mechanism for protein-carbohydrate recognition

The structural and thermodynamic basis for carbohydrate-protein recognition is of considerable importance. NCP-1, which is a component of the Piromyces equi cellulase/hemicellulase complex, presents a provocative model for analyzing how structural and mutational changes can influence the ligand specificity of carbohydrate-binding proteins. NCP-1 contains two "family 29" carbohydrate-binding modules designated CBM29-1 and CBM29-2, respectively, that display unusually broad specificity; the proteins interact weakly with xylan, exhibit moderate affinity for cellulose and mannan, and bind tightly to the beta-1,4-linked glucose-mannose heteropolymer glucomannan. The crystal structure of CBM29-2 in complex with cellohexaose and mannohexaose identified key residues involved in ligand recognition. By exploiting this structural information and the broad specificity of CBM29-2, we have used this protein as a template to explore the evolutionary mechanisms that can lead to significant changes in ligand specificity. Here, we report the properties of the E78R mutant of CBM29-2, which displays ligand specificity that is different from that of wild-type CBM29-2; the protein retains significant affinity for cellulose but does not bind to mannan or glucomannan. Significantly, E78R exhibits a stoichiometry of 0.5 when binding to cellohexaose, and both calorimetry and ultracentrifugation show that the mutant protein displays ligand-mediated dimerization in solution. The three-dimensional structure of E78R in complex with cellohexaose reveals the intriguing molecular basis for this "dimeric" binding mode that involves the lamination of the oligosaccharide between two CBM molecules. The 2-fold screw axis of the ligand is mirrored in the orientation of the two protein domains with adjacent sugar rings stacking against the equivalent aromatic residues in the binding site of each protein molecule of the molecular sandwich. The sandwiching of an oligosaccharide chain between two protein modules, leading to ligand-induced formation of the binding site, represents a completely novel mechanism for protein-carbohydrate recognition that may mimic that displayed by naturally dimeric protein-carbohydrate interactions.     

Analysis of Hsp90 cochaperone interactions reveals a novel mechanism for TPR protein recognition

The chaperone Hsp90 is required for the appropriate regulation of numerous key signaling molecules, including the progesterone receptor (PR). Many important cochaperones bind Hsp90 through their tetratricopeptide repeat (TPR) domains. Two such proteins, GCUNC45 and FKBP52, assist PR chaperoning and are thought to interact sequentially with PR-Hsp90 complexes. TPR proteins bind to the C-terminal MEEVD sequence of Hsp90, but GCUNC45 has been shown also to bind to a novel site near the N-terminus. We now show that FKBP52 is also able to bind to this site, and that these two cochaperones act competitively, through Hsp90, to modulate PR activity. The N-terminal site involves noncontiguous amino acids within or near the ATP binding pocket of Hsp90. TPR interactions at this site are thus strongly regulated by nucleotide binding and Hsp90 conformation. We propose an expanded model for client chaperoning in which the coordinated use of TPR recognition sites at both N- and C-terminal ends of Hsp90 enhances its ability to coordinate interactions with multiple TPR partners.     

Structure-function analysis of the endoplasmic reticulum oxidoreductase TMX3 reveals interdomain stabilization of the N-terminal redox-active domain

Disulfide bond formation in the endoplasmic reticulum is catalyzed by enzymes of the protein disulfide-isomerase family that harbor one or more thioredoxin-like domains. We recently discovered the transmembrane protein TMX3, a thiol-disulfide oxidoreductase of the protein disulfide-isomerase family. Here, we show that the endoplasmic reticulum-luminal region of TMX3 contains three thioredoxin-like domains, an N-terminal redox-active domain (named a) followed by two enzymatically inactive domains (b and b'). Using the recombinantly expressed TMX3 domain constructs a, ab, and abb', we compared structural stability and enzymatic properties. By structural and biophysical methods, we demonstrate that the reduced a domain has features typical of a globular folded domain that is, however, greatly destabilized upon oxidization. Importantly, interdomain stabilization by the b domain renders the a domain more resistant toward chemical denaturation and proteolysis in both the oxidized and reduced form. In combination with molecular modeling studies of TMX3 abb', the experimental results provide a new understanding of the relationship between the multidomain structure of TMX3 and its function as a redox enzyme. Overall, the data indicate that in addition to their role as substrate and co-factor binding domains, redox-inactive thioredoxin-like domains also function in stabilizing neighboring redox-active domains.     

Contributions of the interdomain loop, amino terminus, and subunit interface to the ligand-facilitated dimerization of neurophysin: crystal structures and mutation studies of bovine neurophysin-I

Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55-59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1-6, which interact with the loop and adjacent residues 53-54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1-6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1-6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary.