Ann M. Stock

Histidine kinases and response regulator proteins in two-component signaling systems

Phosphotransfer-mediated signaling pathways allow cells to sense and respond to environmental stimuli. Autophosphorylating histidine protein kinases provide phosphoryl groups for response regulator proteins which, in turn, function as molecular switches that control diverse effector activities. Structural studies of proteins involved in two-component signaling systems have revealed a modular architecture with versatile conserved domains that are readily adapted to the specific needs of individual systems.

Biological Insights from Structures of Two-Component Proteins

Two-component signal transduction based on phosphotransfer from a histidine protein kinase to a response regulator protein is a prevalent strategy for coupling environmental stimuli to adaptive responses in bacteria. In both histidine kinases and response regulators, modular domains with conserved structures and biochemical activities adopt different conformational states in the presence of stimuli or upon phosphorylation, enabling a diverse array of regulatory mechanisms based on inhibitory and/or activating protein-protein interactions imparted by different domain arrangements. This review summarizes some of the recent structural work that has provided insight into the functioning of bacterial histidine kinases and response regulators. Particular emphasis is placed on identifying features that are expected to be conserved among different two-component proteins from those that are expected to differ, with the goal of defining the extent to which knowledge of previously characterized two-component proteins can be applied to newly discovered systems.

Structure of a cholesterol-binding protein deficient in Niemann–Pick type C2 disease

Niemann–Pick disease type C2 (NP-C2) is a fatal hereditary disease characterized by accumulation of low-density lipoprotein-derived cholesterol in lysosomes. Here we report the 1.7-Å resolution crystal structure of the cholesterol-binding protein deficient in this disease, NPC2, and the characterization of its ligand binding properties. Human NPC2 binds the cholesterol analog dehydroergosterol with submicromolar affinity at both acidic and neutral pH. NPC2 has an Ig-like fold stabilized by three disulfide bonds. The structure of the bovine protein reveals a loosely packed region penetrating from the surface into the hydrophobic core that forms adjacent small cavities with a total volume of ≈160 Å3. We propose that this region represents the incipient cholesterol-binding site that dilates to accommodate an ≈740-Å3 cholesterol molecule.

A tale of two components: a novel kinase and a regulatory switch

Histidine protein kinases and response regulators form the basis of phosphotransfer signal transduction pathways. Commonly referred to as two-component systems, these modular and adaptable signaling schemes are prevalent in prokaryotes. Structures of the core domains of histidine kinases reveal a protein kinase fold different from that of the Ser/Thr/Tyr protein kinase family, but similar to that of other ATP binding domains. Recent structure determinations of phosphorylated response regulator domains indicate a conserved mechanism for the propagated conformational change that accompanies phosphorylation of an active site Asp residue. The altered molecular surface promotes specific protein–protein interactions that mediate the downstream response.

Molecular Information Processing: Lessons from Bacterial Chemotaxis

Extracellular information is converted into a usable intracellular form via signal transduction. This critically important process occurs in both unicellular and multicellular organisms throughout the living world and is exhibited by prokaryotic and eukaryotic cells alike. The mechanistic details of information processing in living cells vary from case to case, but diverse systems nevertheless display a number of operating principles in common. Thus, the study of one signaling system can yield insights applicable to others. As a practical matter, the investigation of signal transduction mechanisms in bacteria offers significant technical advantages. This minireview focuses on one of the very best characterized examples of information processing in a biological system, that governing chemotaxis in bacteria, and primarily attempts to summarize what has been learned so far that may be of general interest, as well as point out some features that are not yet fully understood. Many bacteria live in dynamic environments and utilize information processing systems to constantly monitor their surroundings for important changes. Among the appropriate responses to environmental change are alterations in physiology, development, virulence, or location. Although not all bacterial species are motile, many forms of bacterial locomotion exist (often in a single species) including swimming in liquid and gliding, swarming, or twitching over solid surfaces. Organelles known to provide propulsion include flagella and pili; other motors remain unidentified. As a general rule, bacterial species that have invested in a propulsion system are also capable of directed movement, or taxis, to most efficiently translocate to a better environment. Tactic bacteria can respond to a variety of physical stimuli including chemicals, pH, temperature, light, electricity, or magnetism. Genetic investigations in many bacterial species have identified the elements from which logic circuits controlling chemotaxis are constructed. Genome sequencing has revealed the phylogenetic distribution of these signaling elements even in species where chemotaxis has not been studied. As would be expected for any family of signaling pathways built from common components, there are differences in circuit details (e.g. the elements used and the connections between them) among species. However, the central chemotaxis processing pathway appears to be similar across a wide variety of prokaryotic species (both in Bacteria and Archaea), regardless of stimuli or motor type. The most extensive work ( 30 years) has been conducted with Escherichia coli and Salmonella enterica serovar Typhimurium. In these two species, the biochemistry of signaling reactions is well characterized, and atomic resolution structures are available for most individual signaling proteins. Despite immense progress, significant gaps remain in our current state of knowledge about bacterial chemotaxis. What is the nature of higher order interactions that occur between signaling proteins? What is the detailed spatial organization of circuit elements within the cell? What dynamic structural processes occur during signal transmission? What are the structures and functions of those signaling proteins that are phylogenetically widespread but are not present in the best studied model systems? In this minireview, we discuss (i) the circuit elements that comprise chemotaxis information processing systems in bacteria, (ii) how these signaling elements are connected, both logically and physically, and (iii) how these elements communicate information to one another.

Structural Basis of Sterol Binding by NPC2, a Lysosomal Protein Deficient in Niemann-Pick Type C2 Disease

NPC2 is a small lysosomal glycoprotein that binds cholesterol with submicromolar affinity. Deficiency in NPC2 is the cause of Niemann-Pick type C2 disease, a fatal neurovisceral disorder characterized by accumulation of cholesterol in lysosomes. Here we report the crystal structure of bovine NPC2 bound to cholesterol-3-O-sulfate, an analog that binds with greater apparent affinity than cholesterol. Structures of both apo-bound and sterol-bound NPC2 were observed within the same crystal lattice, with an asymmetric unit containing one molecule of apoNPC2 and two molecules of sterol-bound NPC2. As predicted from a previously determined structure of apoNPC2, the sterol binds in a deep hydrophobic pocket sandwiched between the two β-sheets of NPC2, with only the sulfate substituent of the ligand exposed to solvent. In the two available structures of apoNPC2, the incipient ligand-binding pocket, which ranges from a loosely packed hydrophobic core to a small tunnel, is too small to accommodate cholesterol. In the presence of sterol, the pocket expands, facilitated by a slight separation of the β-strands and substantial reorientation of some side chains, resulting in a perfect molding of the pocket around the hydrocarbon portion of cholesterol. A notable feature is the repositioning of two aromatic residues at the tunnel entrance that are essential for NPC2 function. The NPC2 structures provide evidence of a malleable binding site, consistent with the previously documented broad range of sterol ligand specificity.

Sensory transduction in bacterial chemotaxis involves phosphotransfer between CHE proteins

The CheA protein of the Salmonella typhimurium chemotaxis system is phosphorylated by ATP. Phospho-CheA transfers its phosphoryl group to a second chemotaxis protein, CheY. Unlike phospho-CheA, phospho-CheY is relatively unstable, rapidly decaying to phosphate and CheY. We propose that phosphorylation of CheY may play a role in its function as a tumble regulator to control motor behavior in response to attractant and repellent stimuli.

Crystal structure of the chemotaxis receptor methyltransferase CheR suggests a conserved structural motif for binding S-adenosylmethionine.

BACKGROUND Flagellated bacteria swim towards favorable chemicals and away from deleterious ones. The sensing of chemoeffector gradients involves chemotaxis receptors, transmembrane proteins that detect stimuli through their periplasmic domains and transduce signals via their cytoplasmic domains to the downstream signaling components. Signaling outputs from chemotaxis receptors are influenced both by the binding of the chemoeffector ligand to the periplasmic domain and by methylation of specific glutamate residues on the cytoplasmic domain of the receptor. Methylation is catalyzed by CheR, an S-adenosylmethionine-dependent methyltransferase. CheR forms a tight complex with the receptor by binding a region of the receptors that is distinct from the methylation site. CheR belongs to a broad class of enzymes involved in the methylation of a variety of substrates. Until now, no structure from the class of protein methyltransferases has been characterized. RESULTS The structure of the Salmonella typhimurium chemotaxis receptor methyltransferase CheR bound to S-adenosylhomocysteine, a product and inhibitor of the methylation reaction, has been determined at 2.0 A resolution. The structure reveals CheR to be a two-domain protein, with a smaller N-terminal helical domain linked through a single polypeptide connection to a larger C-terminal alpha/beta domain. The C-terminal domain has the characteristics of a nucleotide-binding fold, with an insertion of a small antiparallel beta sheet subdomain. The S-adenosylhomocysteine-binding site is formed mainly by the large domain, with contributions from residues within the N-terminal domain and the linker region. CONCLUSIONS The CheR structure shares some structural similarities with small molecule DNA and RNA methyltransferases, despite a lack of sequence similarity among them. In particular, there is significant structural preservation of the S-adenosylmethionine-binding clefts; the specific length and conformation of a loop in the alpha/beta domain seems to be required for S-adenosylmethionine binding within these enzymes. Unique structural features of CheR, such as the beta subdomain, are probably necessary for CheRs specific interaction with its substrates, the bacterial chemotaxis receptors.

Molecular Strategies for Phosphorylation-Mediated Regulation of Response Regulator Activity

Response regulator (RR) proteins exploit different molecular surfaces in their inactive and active conformations for a variety of regulatory intramolecular and/or intermolecular protein-protein interactions that either inhibit or activate effector domain activities. This versatile strategy enables numerous regulatory mechanisms among RRs. The recent accumulation of structures of inactive and active forms of multidomain RRs and RR complexes has revealed many different domain arrangements that have provided insight into regulatory mechanisms. Although diversity is the rule, even among subfamily members containing homologous domains, several structural modes of interaction and mechanisms of regulation recur frequently. These themes involve interactions at the alpha4-beta5-alpha5 face of the receiver domain, modes of dimerization of receiver domains, and inhibitory or activating heterodomain interactions.

Evidence of Intradomain and Interdomain Flexibility in an OmpR/PhoB Homolog from Thermotoga maritima

Two-component systems, the predominant signal transduction strategy used by prokaryotes, involve phosphorelay from a sensor histidine kinase (HK) to an intracellular response regulator protein (RR) that typically acts as a transcription regulator. RRs are modular proteins, usually composed of a conserved regulatory domain, which functions as a phosphorylation-activated switch, and an attached DNA binding effector domain. The crystal structure of a Thermotoga maritima transcription factor, DrrD, has been determined at 1.5 A resolution, providing the first structural information for a full-length member of the OmpR/PhoB subfamily of RRs. A small interdomain interface occurs between alpha 5 of the regulatory domain and an antiparallel sheet of the effector domain. The lack of an extensive interface in the unphosphorylated protein distinguishes DrrD from other structurally characterized multidomain RRs and suggests a different mode of interdomain regulation.