Signal-sensing triggers the shutdown of HemKR, regulating heme and iron metabolism in the spirochete Leptospira biflexa.

Heme and iron metabolic pathways are highly intertwined, both compounds being essential for key biological processes, yet becoming toxic if overabundant. Their concentrations are exquisitely regulated, including via dedicated two-component systems (TCSs) that sense signals and regulate adaptive responses. HemKR is a TCS present in both saprophytic and pathogenic Leptospira species, involved in the control of heme metabolism.

An allosteric switch ensures efficient unidirectional information transmission by the histidine kinase DesK from .

Phosphorylation carries chemical information in biological systems. In two-component systems (TCSs), the sensor histidine kinase and the response regulator are connected through phosphoryl transfer reactions that may be uni- or bidirectional. Directionality enables the construction of complex regulatory networks that optimize signal propagation and ensure the forward flow of information.

Structural features of sensory two component systems: a synthetic biology perspective.

All living organisms include a set of signaling devices that confer the ability to dynamically perceive and adapt to the fluctuating environment. Two-component systems are part of this sensory machinery that regulates the execution of different genetic and/or biochemical programs in response to specific physical or chemical signals. In the last two decades, there has been tremendous progress in our molecular understanding on how signals are detected, the allosteric mechanisms that control intramolecular information transmission and the specificity determinants that guarantee correct wiring.

Allostery and protein plasticity: the keystones for bacterial signaling and regulation.

Bacteria sense intracellular and environmental signals using an array of proteins as antennas. The information is transmitted from such sensory modules to other protein domains that act as output effectors. Sensor and effector can be part of the same polypeptide or instead be separate diffusible proteins that interact specifically.

The crystal structure of yeast regulatory subunit reveals key evolutionary insights into Protein Kinase A oligomerization.

Protein Kinase A (PKA) is a widespread enzyme that plays a key role in many signaling pathways from lower eukaryotes to metazoans. In mammals, the regulatory (R) subunits sequester and target the catalytic (C) subunits to proper subcellular locations. This targeting is accomplished by the dimerization and docking (D/D) domain of the R subunits.

Protein Dynamics in Phosphoryl-Transfer Signaling Mediated by Two-Component Systems.

The ability to perceive the environment, an essential attribute in living organisms, is linked to the evolution of signaling proteins that recognize specific signals and execute predetermined responses. Such proteins constitute concerted systems that can be as simple as a unique protein, able to recognize a ligand and exert a phenotypic change, or extremely complex pathways engaging dozens of different proteins which act in coordination with feedback loops and signal modulation. To understand how cells sense their surroundings and mount specific adaptive responses, we need to decipher the molecular workings of signal recognition, internalization, transfer, and conversion into chemical changes inside the cell.

Molecular adaptations of NADP-malic enzyme for its function in C photosynthesis in grasses.

In C grasses of agronomical interest, malate shuttled into the bundle sheath cells is decarboxylated mainly by nicotinamide adenine dinucleotide phosphate (NADP)-malic enzyme (C-NADP-ME). The activity of C-NADP-ME was optimized by natural selection to efficiently deliver CO to Rubisco. During its evolution from a plastidic non-photosynthetic NADP-ME, C-NADP-ME acquired increased catalytic efficiency, tetrameric structure and pH-dependent inhibition by its substrate malate.

Two-Component Sensing and Regulation: How Do Histidine Kinases Talk with Response Regulators at the Molecular Level?

Perceiving environmental and internal information and reacting in adaptive ways are essential attributes of living organisms. Two-component systems are relevant protein machineries from prokaryotes and lower eukaryotes that enable cells to sense and process signals. Implicating sensory histidine kinases and response regulator proteins, both components take advantage of protein phosphorylation and flexibility to switch conformations in a signal-dependent way.

FcpB Is a Surface Filament Protein of the Endoflagellum Required for the Motility of the Spirochete .

The spirochete endoflagellum is a unique motility apparatus among bacteria. Despite its critical importance for pathogenesis, the full composition of the flagellum remains to be determined. We have recently reported that FcpA is a novel flagellar protein and a major component of the sheath of the filament of the spirochete .

The crystal structure of the malic enzyme from Candidatus Phytoplasma reveals the minimal structural determinants for a malic enzyme.

Phytoplasmas are wall-less phytopathogenic bacteria that produce devastating effects in a wide variety of plants. Reductive evolution has shaped their genome, with the loss of many genes, limiting their metabolic capacities. Owing to the high concentration of C compounds in plants, and the presence of malic enzyme (ME) in all phytoplasma genomes so far sequenced, the oxidative decarboxylation of L-malate might represent an adaptation to generate energy.

Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography.

We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in , ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR complexes and further purification. The 3D structures of the complex trapped in the phosphotransferase and in the phosphatase reaction steps, were solved by X-ray crystallography using molecular replacement.

Crystallization of FcpA from Leptospira, a novel flagellar protein that is essential for pathogenesis.

The protein FcpA is a unique component of the flagellar filament of spirochete bacteria belonging to the genus Leptospira. Although it plays an essential role in translational motility and pathogenicity, no structures of FcpA homologues are currently available in the PDB. Its three-dimensional structure will unveil the novel motility mechanisms that render pathogenic Leptospira particularly efficient at invading and disseminating within their hosts, causing leptospirosis in humans and animals.

Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action.

Two-component systems (TCS) are protein machineries that enable cells to respond to input signals. Histidine kinases (HK) are the sensory component, transferring information toward downstream response regulators (RR). HKs transfer phosphoryl groups to their specific RRs, but also dephosphorylate them, overall ensuring proper signaling.

Revisiting the human polypeptide GalNAc-T1 and T13 paralogs.

Polypeptide GalNAc-transferases (GalNAc-Ts) constitute a family of 20 human glycosyltransferases (comprising 9 subfamilies), which initiate mucin-type O-glycosylation. The O-glycoproteome is thought to be differentially regulated via the different substrate specificities and expression patterns of each GalNAc-T isoforms. Here, we present a comprehensive in vitro analysis of the peptide substrate specificity of GalNAc-T13, showing that it essentially overlaps with the ubiquitous expressed GalNAc-T1 isoform found in the same subfamily as T13.

A novel flagellar sheath protein, FcpA, determines filament coiling, translational motility and virulence for the Leptospira spirochete.

Leptospira are unique among bacteria based on their helical cell morphology with hook-shaped ends and the presence of periplasmic flagella (PF) with pronounced spontaneous supercoiling. The factors that provoke such supercoiling, as well as the role that PF coiling plays in generating the characteristic hook-end cell morphology and motility, have not been elucidated. We have now identified an abundant protein from the pathogen L.

A coiled coil switch mediates cold sensing by the thermosensory protein DesK.

The thermosensor histidine kinase DesK from Bacillus subtilis senses changes in membrane fluidity initiating an adaptive response. Structural changes in DesK have been implicated in transmembrane signaling, but direct evidence is still lacking. On the basis of structure-guided mutagenesis, we now propose a mechanism of DesK-mediated signal sensing and transduction.

STRUCTURAL VIROLOGY. Conformational plasticity of a native retroviral capsid revealed by x-ray crystallography.

Retroviruses depend on self-assembly of their capsid proteins (core particle) to yield infectious mature virions. Despite the essential role of the retroviral core, its high polymorphism has hindered high-resolution structural analyses. Here, we report the x-ray structure of the native capsid (CA) protein from bovine leukemia virus.

Consecutive interactions with HSP90 and eEF1A underlie a functional maturation and storage pathway of AID in the cytoplasm.

Activation-induced deaminase (AID) initiates mutagenic pathways to diversify the antibody genes during immune responses. The access of AID to the nucleus is limited by CRM1-mediated nuclear export and by an uncharacterized mechanism of cytoplasmic retention. Here, we define a conformational motif in AID that dictates its cytoplasmic retention and demonstrate that the translation elongation factor eukaryotic elongation factor 1 α (eEF1A) is necessary for AID cytoplasmic sequestering.

Allosteric activation of bacterial response regulators: the role of the cognate histidine kinase beyond phosphorylation.

Unlabelled: Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis.

Structural insights into bacterial resistance to cerulenin.

Unlabelled: Cerulenin is a fungal toxin that inhibits both eukaryotic and prokaryotic ketoacyl-acyl carrier protein synthases or condensing enzymes. It has been used experimentally to treat cancer and obesity, and is a potent inhibitor of bacterial growth. Understanding the molecular mechanisms of resistance to cerulenin and similar compounds is thus highly relevant for human health.

Structure of a human IgA1 Fab fragment at 1.55 Å resolution: potential effect of the constant domains on antigen-affinity modulation.

Despite being the most abundant class of immunoglobulins in humans and playing central roles in the adaptive immune response, high-resolution structural data are still lacking for the antigen-binding region of human isotype A antibodies (IgAs). The crystal structures of a human Fab fragment of IgA1 in three different crystal forms are now reported. The three-dimensional organization is similar to those of other Fab classes, but FabA1 seems to be more rigid, being constrained by a hydrophobic core in the interface between the variable and constant domains of the heavy chain (VH-CH1) as well as by a disulfide bridge that connects the light and heavy chains, influencing the relative heavy/light-chain orientation.

Structural and enzymatic insights into the ATP binding and autophosphorylation mechanism of a sensor histidine kinase.

DesK is a sensor histidine kinase (HK) that allows Bacillus subtilis to respond to cold shock, triggering the adaptation of membrane fluidity via transcriptional control of a fatty acid desaturase. It belongs to the HK family HPK7, which includes the nitrogen metabolism regulators NarX/Q and the antibiotic sensor LiaS among other important sensor kinases. Structural information on different HK families is still scarce and several questions remain, particularly concerning the molecular features that determine HK specificity during its catalytic autophosphorylation and subsequent response-regulator phosphotransfer reactions.