A novel signal transduction protein: Combination of solute binding and tandem PAS‐like sensor domains in one polypeptide chain

We report the structural and biochemical characterization of a novel periplasmic ligand‐binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from Lake Retba, in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N‐terminal and a tandem PAS‐like sensor domain at the C‐terminal region. SBP domains are found ubiquitously, and their best known function is in solute transport across membranes. PAS‐like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS‐like domain of the tandem PAS‐like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS‐like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non‐redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.     

The hybrid histidine kinase Slr1759 of the cyanobacterium Synechocystis sp. PCC 6803 contains FAD at its PAS domain

The cyanobacterium Synechocystis sp. PCC 6803 harbours 47 histidine kinases (Hiks). Among these are hybrid histidine kinases with one or two response regulator domains as well as numerous Hiks with several sensory domains. One example is the hybrid histidine kinase Slr1759 (Hik14) that has two PAS domains arranged in tandem linked to a predicted GAF domain. Here, we show that a Slr1759 derivative recombinantly expressed in Escherichia coli has a flavin cofactor. Using truncated Slr1759 variants, it is shown that the flavin associates with the first PAS domain. The cofactor reconstitutes the activity of d-amino acid oxidase apoprotein from pig kidney, indicating that the flavin derivative is FAD. Furthermore, the Slr1759 histidine kinase domain indeed undergoes autophosphorylation in vitro. The phosphorylated product of a recombinant Slr1759 derivative is sensitive to acids, pointing to a histidine residue as the phosphate-accepting group.     

A GAF domain in the hypoxia/NO-inducible Mycobacterium tuberculosis DosS protein binds haem

The majority of the Mycobacterium tuberculosis response to hypoxia and nitric oxide is through the DosRS (DevRS) two-component regulatory system. The N-terminal input domain of the DosS sensor contains two GAF domains. We demonstrate here that the proximal GAF domain binds haem, and identified histidine 149 of DosS as critical to haem-binding; the location of this histidine residue is similar to the cGMP-binding site in a crystal structure of cyclic nucleotide phosphodiesterase 2A. GAF domains are frequently involved in binding cyclic nucleotides, but this is the first GAF domain to be identified that binds haem. In contrast, PAS domains (similar to GAF domains in structure but not primary sequence) frequently use haem cofactors, and these findings further illustrate how the functions of these domains overlap. We propose that the activation of the DosS sensor is controlled through the haem binding of molecular oxygen or nitric oxide.     

C4-dicarboxylates sensing mechanism revealed by the crystal structures of DctB sensor domain

C₄-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C₄-dicarboxylates depend on typical two-component systems (TCS) consisting of a transmembrane sensor histidine kinase and a cytoplasmic response regulator. The DctB-DctD system is the first identified TCS for C₄-dicarboxylates sensing. Direct ligand binding to the sensor domain of DctB is believed to be the first step of the sensing events. In this report, the water-soluble periplasmic sensor domain of Sinorhizobium meliloti DctB (DctBp) was studied, and three crystal structures were solved: the apo protein, a complex with C₄ succinate, and a complex with C₃ malonate. Different from the two structurally known CitA family of carboxylate sensor proteins CitA and DcuS, the structure of DctBp consists of two tandem Per-Arnt-Sim (PAS) domains and one N-terminal helical region. Only the membrane-distal PAS domain was found to bind the ligands, whereas the proximal PAS domain was empty. Comparison of DctB, CitA, and DcuS suggests a detailed stereochemistry of C₄-dicarboxylates ligand perception. The structures of the different ligand binding states of DctBp also revealed a series of conformational changes initiated upon ligand binding and propagated to the N-terminal domain responsible for dimerization, providing insights into understanding the detailed mechanism of the signal transduction of TCS histidine kinases.     

Addition at the Molecular Level: Signal Integration in Designed Per-ARNT-Sim Receptor Proteins

Survival of organisms in dynamic environments requires accurate perception and integration of signals. At the molecular level, signal detection is mediated by signal receptor proteins that largely are of modular composition. Sensor modules, such as the widespread Per-ARNT-Sim (PAS) domains, detect signals and, in response, regulate the biological activity of effector modules. Here, we exploit the modularity of signal receptors to design and engineer synthetic receptors that comprise two PAS sensor domains responsive to different signals, and we use these signals to control the activity of a histidine kinase effector. Designed two-input PAS receptors detected oxygen and blue light in a positive cooperative manner. The extent of the response to the signals was dictated by domain topology: the dominant regulatory effect was exerted by the PAS domain proximal to the effector domain. The presence of one sensor domain modulated the signal response function of the other. Sequence and structural data on natural receptors with tandem PAS domains show that these are predominantly linked by short amphipathic α-helices. Signals from multiple sensor domains could be integrated and propagated to the effector domain as torques. Our results inform the rational design of receptors that integrate multiple signals to modulate cellular behavior.     

Structural and functional modularity of proteins in the de novo purine biosynthetic pathway

It is generally accepted that naturally existing functional domains can serve as building blocks for complex protein structures, and that novel functions can arise from assembly of different combinations of these functional domains. To inform our understanding of protein evolution and explore the modular nature of protein structure, two model enzymes were chosen for study, purT‐encoded glycinamide ribonucleotide formyltransferase (PurT) and purK‐encoded N⁵‐carboxylaminoimidazole ribonucleotide synthetase (PurK). Both enzymes are found in the de novo purine biosynthetic pathway of Escherichia coli. In spite of their low sequence identity, PurT and PurK share significant similarity in terms of tertiary structure, active site organization, and reaction mechanism. Their characteristic three domain structures categorize both PurT and PurK as members of the ATP‐grasp protein superfamily. In this study, we investigate the exchangeability of individual protein domains between these two enzymes and the in vivo and in vitro functional properties of the resulting hybrids. Six domain‐swapped hybrids were unable to catalyze full wild‐type reactions, but each hybrid protein could catalyze partial reactions. Notably, an additional loop replacement in one of the domain‐swapped hybrid proteins was able to restore near wild‐type PurK activity. Therefore, in this model system, domain‐swapped proteins retained the ability to catalyze partial reactions, but further modifications were required to efficiently couple the reaction intermediates and achieve catalysis of the full reaction. Implications for understanding the role of domain swapping in protein evolution are discussed.     

The crystal structure of Arabidopsis BON1 provides insights into the copine protein family

The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium‐dependent membrane‐binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure. The first structure of this protein family has been determined to 2.5‐Å resolution and shows the structural features of the three conserved domains C2A, C2B and vWA. The structure reveals the third Ca²⁺‐binding region in C2A domain is longer than classical C2 domains and a novel Ca²⁺ binding site in the vWA domain. The structure of BON1 bound to Mn²⁺ is also presented. The binding of the C2 domains to phospholipid (PSF) has been modeled and provides an insight into the lipid‐binding mechanism of the copine proteins. Furthermore, the selectivity of the separate C2A and C2B domains and intact BON1 to bind to different phospholipids has been investigated, and we demonstrated that BON1 could mediate aggregation of liposomes in response to Ca²⁺. These studies have formed the basis of further investigations into the important role that the copine proteins play in vivo.     

Biochemical Characterization of an Adenylate Cyclase, CyaB1, in the Cyanobacterium Anabaena sp. Strain PCC 7120

cyaB1 gene encodes a novel type of adenylate cyclase. The catalytic domain is located in the carboxyl-terminal half, while the GAF and PAS domains are conserved in the amino-terminal half. Recombinant CyaB1 and a truncated CyaB1 lacking the amino-terminal domain (ΔN–CyaB1) were purified and characterized. The purified CyaB1 is activated by divalent cations, such as Mg²⁺ and Mn²⁺, like other types of adenylate cyclase. The activity of CyaB1 was slightly elevated by forskolin, but was not affected by cGMP, irrespective of the presence of the cGMP binding motif in the GAF domain. The specific activity of ΔN–CyaB1 is one-eighteenth that of CyaB1, whereas the Km values of both proteins are almost the same. The results suggest that the amino-terminal half has a positive regulatory effect on the catalytic activity. Received 27 April 2001/ Accepted in revised form 6 July 2001     

The activity of the RGA5 sensor NLR from rice requires binding of its integrated HMA domain to effectors but not HMA domain self-interaction

The rice nucleotide-binding (NB) and leucine-rich repeat (LRR) domain immune receptors (NLRs) RGA4 and RGA5 form a helper NLR/sensor NLR (hNLR/sNLR) pair that specifically recognizes the effectors AVR-Pia and AVR1-CO39 from the blast fungus Magnaporthe oryzae. While RGA4 contains only canonical NLR domains, RGA5 has an additional unconventional heavy metal-associated (HMA) domain integrated after its LRR domain. This RGA5_HMA domain binds the effectors and is crucial for their recognition. Investigation of the three-dimensional structure of the AVR1-CO39/RGA5_HMA complex by X-ray crystallography identified a candidate surface for effector binding in the HMA domain and showed that the HMA domain self-interacts in the absence of effector through the same surface. Here, we investigated the relevance of this HMA homodimerization for RGA5 function and the role of the RGA5_HMA effector-binding and self-interaction surface in effector recognition. By analysing structure-informed point mutations in the RGA5_HMA-binding surface in protein interaction studies and in Nicotiana benthamiana cell death assays, we found that HMA self-interaction does not contribute to RGA5 function. However, the effector-binding surface of RGA5_HMA identified by X-ray crystallography is crucial for both in vitro and in vivo effector binding as well as effector recognition. These results support the current hypothesis that noncanonical integrated domains of NLRs act primarily as effector traps and deepen our understanding of the sNLRs' function within NLR pairs.     

Influence of PAS Domain Flanking Regions on Oligomerisation and Redox Signalling By NifL

Per-ARNT-Sim (PAS) domains constitute a typically dimeric, conserved α/β tertiary fold of approximately 110 amino acids that perform signalling roles in diverse proteins from all kingdoms of life. The amino terminal PAS1 domain of NifL from Azotobacter vinelandii accommodates a redox-active FAD group; elevation of cytosolic oxygen concentrations result in FAD oxidation and a concomitant conformational re-arrangement that is relayed via a short downstream linker to a second PAS domain, PAS2. At PAS2, the signal is amplified and passed on to effector domains generating the ‘on’ (inhibitory) state of the protein. Although the crystal structure of oxidised PAS1 reveals regions that contribute to the dimerisation interface, 21 amino acids at the extreme N-terminus of NifL, are unresolved. Furthermore, the structure and function of the linker between the two PAS domains has not been determined. In this study we have investigated the importance to signalling of residues extending beyond the core PAS fold. Our results implicate the N-terminus of PAS1 and the helical linker connecting the two PAS domains in redox signal transduction and demonstrate a role for these flanking regions in controlling the oligomerisation state of PAS1 in solution.     

Accurate prediction of interfacial residues in two‐domain proteins using evolutionary information: Implications for three‐dimensional modeling

With the preponderance of multidomain proteins in eukaryotic genomes, it is essential to recognize the constituent domains and their functions. Often function involves communications across the domain interfaces, and the knowledge of the interacting sites is essential to our understanding of the structure–function relationship. Using evolutionary information extracted from homologous domains in at least two diverse domain architectures (single and multidomain), we predict the interface residues corresponding to domains from the two‐domain proteins. We also use information from the three‐dimensional structures of individual domains of two‐domain proteins to train naïve Bayes classifier model to predict the interfacial residues. Our predictions are highly accurate (～85%) and specific (～95%) to the domain–domain interfaces. This method is specific to multidomain proteins which contain domains in at least more than one protein architectural context. Using predicted residues to constrain domain–domain interaction, rigid‐body docking was able to provide us with accurate full‐length protein structures with correct orientation of domains. We believe that these results can be of considerable interest toward rational protein and interaction design, apart from providing us with valuable information on the nature of interactions. Proteins 2014; 82:1219–1234. © 2013 Wiley Periodicals, Inc.     

Structure and function of the juxtamembrane GAF domain of potassium biosensor KdpD

KdpD/KdpE two‐component signaling system regulates expression of a high affinity potassium transporter responsible for potassium homeostasis. The C‐terminal module of KdpD consists of a GAF domain linked to a histidine kinase domain. Whereas certain GAF domains act as regulators by binding cyclic nucleotides, the role of the juxtamembrane GAF domain in KdpD is unknown. We report the high‐resolution crystal structure of KdpD GAF domain (KdpD^G) consisting of five α‐helices, four β‐sheets and two large loops. KdpD^G forms a symmetry‐related dimer, wherein parallelly arranged monomers contribute to a four‐helix bundle at the dimer‐interface, SAXS analysis of KdpD C‐terminal module reveals an elongated structure that is a dimer in solution. Substitution of conserved residues with various residues that disrupt the dimer interface produce a range of effects on gene expression demonstrating the importance of the interface in inactive to active transitions during signaling. Comparison of ligand binding site of the classic cyclic nucleotide‐binding GAF domains to KdpD^G reveals structural differences arising from naturally occurring substitutions in primary sequence of KdpD^G that modifies the canonical NKFDE sequence motif required for cyclic nucleotide binding. Together these results suggest a structural role for KdpD^G in dimerization and transmission of signal to the kinase domain.     

Close encounters of the third kind: disordered domains and the interactions of proteins

Protein–protein interactions are thought to be mediated by domains, which are autonomous folding units of proteins. Recently, a second type of interaction has been suggested, mediated by short segments termed linear motifs, which are related to recognition elements of intrinsically disordered regions. Here, we propose a third kind of protein–protein recognition mechanism, mediated by disordered regions longer than 20–30 residues. Bioinformatics predictions and well‐characterized examples, such as the kinase‐inhibitory domain of Cdk inhibitors and the Wiskott–Aldrich syndrome protein (WASP)‐homology domain 2 of actin‐binding proteins, show that these disordered regions conform to the definition of domains rather than motifs, i.e., they represent functional, evolutionary, and structural units. Their functions are distinct from those of short motifs and ordered domains, and establish a third kind of interaction principle. With these points, we argue that these long disordered regions should be recognized as a distinct class of biologically functional protein domains.     

Structures of domains I and IV from YbbR are representative of a widely distributed protein family

YbbR domains are widespread throughout Eubacteria and are expressed as monomeric units, linked in tandem repeats or cotranslated with other domains. Although the precise role of these domains remains undefined, the location of the multiple YbbR domain‐encoding ybbR gene in the Bacillus subtilis glmM operon and its previous identification as a substrate for a surfactin‐type phosphopantetheinyl transferase suggests a role in cell growth, division, and virulence. To further characterize the YbbR domains, structures of two of the four domains (I and IV) from the YbbR‐like protein of Desulfitobacterium hafniense Y51 were solved by solution nuclear magnetic resonance and X‐ray crystallography. The structures show the domains to have nearly identical topologies despite a low amino acid identity (23%). The topology is dominated by β‐strands, roughly following a “figure 8” pattern with some strands coiling around the domain perimeter and others crossing the center. A similar topology is found in the C‐terminal domain of two stress‐responsive bacterial ribosomal proteins, TL5 and L25. Based on these models, a structurally guided amino acid alignment identifies features of the YbbR domains that are not evident from naïve amino acid sequence alignments. A structurally conserved cis‐proline (cis‐Pro) residue was identified in both domains, though the local structure in the immediate vicinities surrounding this residue differed between the two models. The conservation and location of this cis‐Pro, plus anchoring Val residues, suggest this motif may be significant to protein function.