The Neurospora crassa photoreceptor Vivid tunes blue-light responses and modulates gating of the circadian clock. Crystal structures of dark-state and light-state Vivid reveal a light, oxygen, or voltage Per-Arnt-Sim domain with an unusual N-terminal cap region and a loop insertion that accommodates the flavin cofactor. Photoinduced formation of a cystein-flavin adduct drives flavin protonation to induce an N-terminal conformational change. A cysteine-to-serine substitution remote from the flavin adenine dinucleotide binding site decouples conformational switching from the flavin photocycle and prevents Vivid from sending signals in Neurospora. Key elements of this activation mechanism are conserved by other photosensors such as White Collar-1, ZEITLUPE, ENVOY, and flavin-binding, kelch repeat, F-BOX 1 (FKF1).The PAS (Per-Arnt-Sim) protein superfamily transduces signals from diverse biological cues, often by coupling cofactor chemistry to alterations in protein conformation or association (1). The canonical PAS domain protein photoactive yellow protein (PYP) and the light, oxygen, or voltage (LOV) PAS subclass sense blue light in bacteria, plants, and fungi (2, 3). Despite extensive photochemical and structural characterization of such bluelight sensors (2, 4-8), the mechanism by which cofactor excitation leads to biological signal propagation remains an open question.The filamentous fungus Neurospora crassa employs two blue-light sensors with LOV domains, White Collar-1 (WC-1) and Vivid (VVD) to regulate a variety of light responses (9). WC-1 and nonphotosensitive WC-2 form a complex (WCC) that resets the circadian clock by activating transcription of the clock oscillator protein Frequency (FRQ), as well as many other genes (9, 10). VVD, a small PAS protein devoid of auxiliary domains, tunes Neurospora's blue-light response by attenuating activation of the WCC. VVD is essential for response to changing levels of light and for adaptation under constant light (11)(12)(13)(14). VVD and WC-1 share sequence similarity in a core LOV domain and surrounding regions (15). Swapping the WC-1 core LOV domain with that from VVD maintains some light responses in Neurospora (16). VVD and WC-1 require flavin adenine dinucleotide (FAD) for activity instead of flavin mononucleotide (FMN), which is used by plant and algal LOV-containing proteins known as phototropins (9,12,17,18).We report the crystal structure of VVD in its dark-and light-adapted states and show how chemical changes at the active center generate conformational change at the N terminus of

Histidine kinases allow bacteria, plants, and fungi to sense and respond to their environment. The 2.6 A resolution crystal structure of Thermotoga maritima CheA (290-671) histidine kinase reveals a dimer where the functions of dimerization, ATP binding, and regulation are segregated into domains. The kinase domain is unlike Ser/Thr/Tyr kinases but resembles two ATPases, Gyrase B and Hsp90. Structural analogies within this superfamily suggest that the P1 domain of CheA provides the nucleophilic histidine and activating glutamate for phosphotransfer. The regulatory domain, which binds the homologous receptor-coupling protein CheW, topologically resembles two SH3 domains and provides different protein recognition surfaces at each end. The dimerization domain forms a central four-helix bundle about which the kinase and regulatory domains pivot on conserved hinges to modulate transphosphorylation. Different subunit conformations suggest that relative domain motions link receptor response to kinase activity.

Chemoreceptor arrays are supramolecular transmembrane machines of unknown structure that allow bacteria to sense their surroundings and respond by chemotaxis. We have combined X-ray crystallography of purified proteins with electron cryotomography of native arrays inside cells to reveal the arrangement of the component transmembrane receptors, histidine kinases (CheA) and CheW coupling proteins. Trimers of receptor dimers lie at the vertices of a hexagonal lattice in a "two-facing-two" configuration surrounding a ring of alternating CheA regulatory domains (P5) and CheW couplers. Whereas the CheA kinase domains (P4) project downward below the ring, the CheA dimerization domains (P3) link neighboring rings to form an extended, stable array. This highly interconnected protein architecture underlies the remarkable sensitivity and cooperative nature of transmembrane signaling in bacterial chemotaxis.protein structure | hybrid methods | two-component systems C hemotactic bacteria sense their surrounding conditions through an array of transmembrane chemoreceptors (methylaccepting chemotaxis proteins, or MCPs), which are found with histidine kinases (CheA) and couplers (CheW) in polar clusters (1-3) and along the sides of cells (4, 5). Repellents and attractants bind to the periplasmic domains of the MCPs either directly (6, 7) or via periplasmic binding proteins (8). The status of the binding domain is transmitted along the length of the receptors through the transmembrane region, across one or more HAMP (histidine kinases, adenyl cyclases, MCPs, and some phosphatases) domain (s), and down the coiled-coil cytoplasmic signaling domain where they ultimately regulate the activity of the histidine kinase CheA located at the receptors' cytoplasmic tips (1-3, 9). CheA is a large, five-domain (P1-P5) protein. P1 contains the substrate histidine, P2 is the docking site for the response regulator CheY, P3 is the dimerization domain, P4 binds ATP and is the kinase, and P5 binds CheW. P1, P2, and P3 are connected to each other by flexible linkers (1, 2). Crystal structures of all domains from Thermotoga maritima CheA are already available (10-13).In the model system Escherichia coli, the addition of attractants or removal of repellents results in kinase inactivation, causing the flagella to rotate counterclockwise. In that case, the multiple flagella form one large bundle that propels the cells smoothly forward and the cells "run." In contrast, addition of repellents or removal of attractants activates CheA, which autophosphorylates and then transfers the phosphoryl group to the second messenger CheY, which in turn binds to the flagellar motors and changes the direction of flagellar rotation to clockwise (CW). This switch results in disassembly of the flagellar bundle and causes the cells to "tumble" (14). CheA also regulates the activity of the receptor-modifying enzyme CheB (a methylesterase), which together with CheR (a methyltransferase) controls the methylation state of residues in the MCP adaptation region (1). Methylation...

In bacterial chemotaxis, an assembly of transmembrane receptors, the CheA histidine kinase and the adaptor protein CheW processes environmental stimuli to regulate motility. The structure of a Thermotoga maritima receptor cytoplasmic domain defines CheA interaction regions and metal ion-coordinating charge centers that undergo chemical modification to tune receptor response. Dimeric CheA-CheW, defined by crystallography and pulsed ESR, positions two CheWs to form a cleft that is lined with residues important for receptor interactions and sized to clamp one receptor dimer. CheW residues involved in kinase activation map to interfaces that orient the CheW clamps. CheA regulatory domains associate in crystals through conserved hydrophobic surfaces. Such CheA self-contacts align the CheW receptor clamps for binding receptor tips. Linking layers of ternary complexes with close-packed receptors generates a lattice with reasonable component ratios, cooperative interactions among receptors and accessible sites for modification enzymes.

Receptor-like protein-tyrosine phosphatases (RPTPs), like their non-receptor counterparts, regulate the level of phosphotyrosine-containing proteins derived from the action of protein-tyrosine kinases. RPTPs are type-I integral membrane proteins which contain one or two catalytic domains in their cytoplasmic region. It is not known whether extracellular ligands regulate the activity of RPTPs. Here we describe the crystal structure of the membrane-proximal catalytic domain (D1) of a typical RPTP, murine RPTP alpha. Significant structural deviations from the PTP1B fold reside within the amino-terminal helix-turn-helix segment of RPTPalphaD1 (residues 214 to 242) and a distinctive two-stranded beta-sheet formed between residues 211-213 and 458-461. The turn of the N-terminal segment inserts into the active site of a dyad-related D1 monomer. On the basis of two independent crystal structures, sequence alignments, and the reported biological activity of EGF receptor/CD45 chimaeras, we propose that dimerization and active-site blockage is a physiologically important mechanism for downregulating the catalytic activity of RPTPalpha and other RPTPs.

Summary HAMP domains are widespread prokaryotic signaling modules found as single domains or poly-HAMP chains in both transmembrane and soluble proteins. The crystal structure of a 3 unit poly-HAMP chain from the P. aeruginosa soluble receptor Aer2 defines a universal parallel four-helix bundle architecture for diverse HAMP domains. Two contiguous domains integrate to form a concatenated di-HAMP structure. The three HAMP domains display two distinct conformations that differ by changes in helical register, crossing angles, and rotation. These conformations are stabilized by different subsets of conserved residues. Known signals delivered to HAMP would be expected to switch the relative stability of the two conformations and the position of a coiled-coil phase stutter at the junction with downstream helices. We propose that the two conformations represent opposing HAMP signaling states and suggest a signaling mechanism whereby HAMP domains interconvert between the two states, which alternate down a poly-HAMP chain.

Bacterial chemosensory arrays are composed of extended networks of chemoreceptors (also known as methyl-accepting chemotaxis proteins, MCPs), the histidine kinase CheA, and the adaptor protein CheW. Models of these arrays have been developed from cryoelectron microscopy, crystal structures of binary and ternary complexes, NMR spectroscopy, mutational data and biochemical studies. A new 3.2 Å resolution crystal structure of a T. maritima MCP protein interaction region in complex with the CheA kinase-regulatory module (P4–P5) and adaptor protein CheW provides sufficient detail to define residue contacts at the interfaces formed among the three proteins. As in a previous 4.5 Å resolution structure, CheA-P5 and CheW interact through conserved hydrophobic surfaces at the ends of their β-barrels to from pseudo six-fold symmetric rings in which the two proteins alternate around the circumference. The interface between P5 subdomain 1 and CheW subdomain 2 was anticipated from previous studies, whereas the related interface between CheW subdomain 1 and P5 subdomain 2 has only been observed in these ring assemblies. The receptor forms an unexpected structure in that the helical hairpin tip of each subunit has “unzipped” into a continuous α-helix; four such helices associate into a bundle and the tetramers bridge adjacent P5-CheW rings in the lattice through interactions with both P5 and CheW. P5 and CheW each bind a receptor helix with a groove of conserved hydrophobic residues between subdomains 1 and 2. P5 binds the receptor helix N-terminal to the tip region (lower site), whereas CheW binds the same helix with inverted polarity near the bundle end (upper site). Sequence comparisons among different evolutionary classes of chemotaxis proteins show that the binding partners undergo correlated changes at key residue positions that involve the lower site. Such evolutionary analyses argue that both CheW and P5 bind to the receptor tip at overlapping positions. Computational genomics further reveal that two distinct CheW proteins in Thermotogae utilize the analogous recognition motifs to couple different receptor classes to the same CheA kinase. Important residues for function previously identified by mutagenesis, chemical modification and biophysical approaches also map to these same interfaces. Thus, although the native CheW-receptor interaction is not observed in the present crystal structure, the bioinformatics and previous data predict key features of this interface. The companion study of the P5-receptor interface in native arrays (Piasta et al. (2013) Biochemistry; Companion paper) shows that, despite the non-native receptor fold in the present crystal structure, the local helix-in-groove contacts of the crystallographic P5-receptor interaction are present in native arrays and are essential for receptor regulation of kinase activity.

The function and regulation of the receptorlike transmembrane protein tyrosine phosphatases (RPTPs) are not well understood. Ligand-induced dimerization inhibited the function of the epidermal growth factor receptor (EGFR)-RPTP CD45 chimera (EGFR-CD45) in T cell signal transduction. Properties of mutated EGFR-CD45 chimeras supported a general model for the regulation of RPTPs, derived from the crystal structure of the RPTPalpha membrane-proximal phosphatase domain. The phosphatase domain apparently forms a symmetrical dimer in which the catalytic site of one molecule is blocked by specific contacts with a wedge from the other.