Light-oxygen-voltage (LOV) domains sense blue light through the photochemical formation of a cysteinyl-flavin covalent adduct. Concurrent protonation at the flavin N5 position alters the hydrogen bonding interactions of an invariant Gln residue that has been proposed to flip its amide side chain as a critical step in the propagation of conformational change. Traditional molecular dynamics (MD) and replica-exchange MD (REMD) simulations of the well-characterized LOV protein Vivid (VVD) demonstrate that the Gln182 amide indeed reorients by ∼180° in response to either adduct formation or reduction of the isoalloxazine ring to the neutral semiquinone, both of which involve N5 protonation. Free energy simulations reveal that the relative free energies of the flipped Gln conformation and the flipping barrier are significantly lower in the light-adapted state. The Gln182 flip stabilizes an important hinge-bβ region between the PAS β-sheet and the N-terminal cap helix that in turn destabilizes an N-terminal latch region against the PAS core. Release of the latch, observed both experimentally and in the simulations, is known to mediate light-induced VVD dimerization. This computational study of a LOV protein, unprecedented in its agreement with experiment, provides an atomistic view of long-range allosteric coupling in a photoreceptor.

HAMP domains are dimeric, four-helix bundles that transduce conformational signals in bacterial receptors. Genetic studies of the Escherichia coli serine receptor (Tsr) provide an opportunity to understand HAMP conformational behavior in terms of functional output. To increase its stability, the Tsr HAMP domain was spliced into a poly-HAMP unit from the Pseudomonas aeruginosa Aer2 receptor. Within the chimera, the Tsr HAMP undergoes a thermal melting transition at a temperature much lower than that of the Aer2 HAMP domains. Pulse-dipolar electron spin resonance spectroscopy and site-specific spin-labeling confirm that the Tsr HAMP maintains a four-helix bundle. Pulse-dipolar electron spin resonance spectroscopy was also used to study three well-characterized HAMP mutational phenotypes: those that cause flagella rotation that is counterclockwise (CCW) A and kinase-off; CCW B and also kinase-off; and, clockwise (CW) and kinase-on. Conformational properties of the three HAMP variants support a biphasic model of dynamic bundle stability, but also indicate distinct conformational changes within the helix bundle. Functional kinase-on (CW) and kinase-off (CCW A) states differ by concerted changes in the positions of spin-label sites at the base of the bundle. Opposite shifts in the subunit separation distances of neighboring residues at the C-termini of the α1 and α2 helices are consistent with a helix scissors motion or a gearbox rotational model of HAMP activation. In the drastic kinase-off lesion of CCW B, the α1 helices unfold and the α2 helices form a tight two-helix coiled-coil. The substitution of a critical residue in the Tsr N-terminal linker or control cable reduces conformational heterogeneity at the N-terminus of α1 but does not affect structure at the C-terminus of α2. Overall, the data suggest that transitions from on- to off-states involve decreased motional amplitudes of the Tsr HAMP coupled with helix rotations and movements toward a two-helix packing mode.

The tryptophan 191 cation radical of cytochrome c peroxidase (CcP) compound I (Cpd I) mediates long-range electron transfer (ET) to cytochrome c (Cc). Here we test the effects of chemical substitution at position 191. CcP W191Y forms a stable tyrosyl radical upon reaction with peroxide and produces spectral properties similar to those of Cpd I but has low reactivity toward reduced Cc. CcP W191G and W191F variants also have low activity, as do redox ligands that bind within the W191G cavity. Crystal structures of complexes between Cc and CcP W191X (X = Y, F, or G), as well as W191G with four bound ligands reveal similar 1:1 association modes and heme pocket conformations. The ligands display structural disorder in the pocket and do not hydrogen bond to Asp235, as does Trp191. Well-ordered Tyr191 directs its hydroxyl group toward the porphyrin ring, with no basic residue in the range of interaction. CcP W191X (X = Y, F, or G) variants substituted with zinc-porphyrin (ZnP) undergo photoinduced ET with Cc(III). Their slow charge recombination kinetics that result from loss of the radical center allow resolution of difference spectra for the charge-separated state [ZnP+, Cc(II)]. The change from a phenyl moiety at position 191 in W191F to a water-filled cavity in W191G produces effects on ET rates much weaker than the effects of the change from Trp to Phe. Low net reactivity of W191Y toward Cc(II) derives either from the inability of ZnP+ or the Fe-CcP ferryl to oxidize Tyr or from the low potential of the resulting neutral Tyr radical.

Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa. Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions.

Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to form extended membrane arrays that receive and transduce environmental signals. A receptor trimers-of-dimers resides at each vertex of the hexagonal protein lattice. CheA is fully activated and regulated when it is integrated into the receptor assembly. To mimic these states in solution, we have engineered chemoreceptor cytoplasmic kinase-control modules (KCMs) based on the Escherichia coli aspartate receptor Tar that are covalently fused and trimerized by a foldon domain (TarFO). Small-angle X-ray scattering, multi-angle light scattering, and pulsed-dipolar electron spin resonance spectroscopy of spin-labeled proteins indicate that the TarFO modules assemble into homogeneous trimers wherein the protein interaction regions closely associate at the end opposite to the foldon domains. The TarFO variants greatly increase the saturation levels of phosphorylated CheA (CheA-P), indicating that the association with a trimer of receptor dimers changes the fraction of active kinase. However, the rate constants for CheA-P formation with the Tar variants are low compared to those for autophosphorylation by free CheA, and net phosphotransfer from CheA to CheY does not increase commensurately with CheA autophosphorylation. Thus, the Tar variants facilitate slow conversion to an active form of CheA that then undergoes stable autophosphorylation and is capable of subsequent phosphotransfer to CheY. Free CheA is largely incapable of phosphorylation but contains a small active fraction. Addition of TarFO to CheA promotes a planar conformation of the regulatory domains consistent with array models for the assembly state of the ternary complex and different from that observed with a single inhibitory receptor. Introduction of TarFO into E. coli cells activates endogenous CheA to produce increased clockwise flagellar rotation, with the effects increasing in the presence of the chemotaxis methylation system (CheB/CheR). Overall, the TarFO modules demonstrate that trimerized signaling tips self-associate, bind CheA and CheW, and facilitate conversion of CheA to an active conformation.

Dynamics are hypothesized to play an important role in the transmission of signals across membranes by receptors. Bacterial chemoreceptors are long helical proteins that consist of a periplasmic ligand-binding domain; a transmembrane region; a cytoplasmic HAMP (histidine kinase, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) domain; and a kinase-control module (KCM). The KCM is further composed of adaptation, hinge, and protein interaction regions (PIRs), the latter of which binds the histidine kinase CheA and adaptor CheW. Fusions of the Escherichia coli aspartate receptor KCM to HAMP domains of defined structure (H1-Tar vs. H1-2-Tar) give opposite responses in phosphotransfer and cellular assays, despite similar binding to CheA and CheW. Pulsed dipolar ESR spectroscopy (PDS) of these isolated on and off dimeric effectors reveals that, in the kinase-on state, the HAMP is more conformationally destabilized compared with the PIR, whereas in the kinase-off state, the HAMP is more compact, and the PIR samples a greater breadth of conformations. On and off HAMP states produce different conformational effects at the KCM junction, but these differences decrease through the adaptation region and into the hinge only to return with the inverted relationship in the PIR. Continuous wave–ESR of the spin-labeled proteins confirms that broader PDS distance distributions correlate with increased rates of dynamics. Conformational breadth in the adaptation region changes with charge alterations caused by modification enzymes. Activating modifications broaden the HAMP conformational ensemble but correspondingly, compact the PIR. Thus, chemoreceptors behave as coupled units, in which dynamics in regions proximal and distal to the membrane change coherently but with opposite sign.

Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and NO in a two-step process involving the intermediate Nω-hydroxy-l-arginine (NHA). It was shown that Cpd I is the oxygenating species for l-arginine; the hydroperoxo ferric intermediate is the reactive intermediate with NHA. Methylation of the Nω-OH and Nω-H of NHA significantly inhibits the conversion of NHA into NO and l-citrulline by mammalian NOS. Kinetic studies now show that Nω-methylation of NHA has a qualitatively similar effect on H2O2-dependent catalysis by bacterial gsNOS. To elucidate the effect of methylating Nω-hydroxy l-arginine on the properties and reactivity of the one-electron-reduced oxy-heme center of NOS, we have applied cryoreduction/annealing/EPR/ENDOR techniques. Measurements of solvent kinetic isotope effects during 160 K cryoannealing cryoreduced oxy-gsNOS/NHA confirm the hydroperoxo ferric intermediate as the catalytically active species of step two. Product analysis for cryoreduced samples with methylated NHA’s, NHMA, NMOA, and NMMA, annealed to 273 K, show a correlation of yields of l-citrulline with the intensity of the g 2.26 EPR signal of the peroxo ferric species trapped at 77 K, which converts to the reactive hydroperoxo ferric state. There is also a correlation between the yield of l-citrulline in these experiments and kobs for the H2O2-dependent conversion of the substrates by gsNOS. Correspondingly, no detectable amount of cyanoornithine, formed when Cpd I is the reactive species, was found in the samples. Methylation of the NHA guanidinium Nω-OH and Nω-H inhibits the second NO-producing reaction by favoring protonation of the ferric-peroxo to form unreactive conformers of the ferric-hydroperoxo state. It is suggested that this is caused by modification of the distal-pocket hydrogen-bonding network of oxy gsNOS and introduction of an ordered water molecule that facilitates delivery of the proton(s) to the one-electron-reduced oxy-heme moiety. These results illustrate how variations in the properties of the substrate can modulate the reactivity of a monooxygenase.

Light–oxygen–voltage (LOV) domains bind a flavin chromophore to serve as blue light sensors in a wide range of eukaryotic and prokaryotic proteins. LOV domains are associated with a variable effector domain or a separate protein signaling partner to execute a wide variety of functions that include regulation of kinases, generation of anti-sigma factor antagonists, and regulation of circadian clocks. Here we present the crystal structure, photocycle kinetics, association properties, and spectroscopic features of a full-length LOV domain protein from Rhodobacter sphaeroides (RsLOV). RsLOV exhibits N- and C-terminal helical extensions that form an unusual helical bundle at its dimer interface with some resemblance to the helical transducer of sensory rhodopsin II. The blue light-induced conformational changes of RsLOV revealed from a comparison of light- and dark-state crystal structures support a shared signaling mechanism of LOV domain proteins that originates with the light-induced formation of a flavin–cysteinyl photoadduct. Adduct formation disrupts hydrogen bonding in the active site and propagates structural changes through the LOV domain core to the N- and C-terminal extensions. Single-residue variants in the active site and dimer interface of RsLOV alter photoadduct lifetimes and induce structural changes that perturb the oligomeric state. Size exclusion chromatography, multiangle light scattering, small-angle X-ray scattering, and cross-linking studies indicate that RsLOV dimerizes in the dark but, upon light excitation, dissociates into monomers. This light-induced switch in oligomeric state may prove to be useful for engineering molecular associations in controlled cellular settings.

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 Thermotoga 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 form pseudo 6-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 (accompanying paper Piasta et al. (2013) Biochemistry, DOI: 10.1021/bi400385c) 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.

Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation.

Light-induced conformational changes in a blue-light sensor facilitate its dimerization.

The signaling apparatus that controls bacterial chemotaxis is composed of a core complex containing chemoreceptors, the histidine autokinase CheA, and the coupling protein CheW. Site-specific spin labeling and pulsed dipolar ESR spectroscopy (PDS) have been applied to investigate the structure of a soluble ternary complex formed by Thermotoga maritima CheA (TmCheA), CheW, and receptor signaling domains. Thirty-five symmetric spin-label sites (SLSs) were engineered into the five domains of the CheA dimer and CheW to provide distance restraints within the CheA:CheW complex in the absence and presence of a soluble receptor that inhibits kinase activity (Tm14). Additional PDS restraints among spin-labeled CheA, CheW, and an engineered single-chain receptor labeled at six different sites allow docking of the receptor structure relative to the CheA:CheW complex. Disulfide cross-linking between selectively incorporated Cys residues finds two pairs of positions that provide further constraints within the ternary complex: one involving Tm14 and CheW and another involving Tm14 and CheA. The derived structure of the ternary complex indicates a primary site of interaction between CheW and Tm14 that agrees well with previous biochemical and genetic data for transmembrane chemoreceptors. The PDS distance distributions are most consistent with only one CheW directly engaging one dimeric Tm14. The CheA dimerization domain (P3) aligns roughly antiparallel to the receptor-conserved signaling tip but does not interact strongly with it. The angle of the receptor axis with respect to P3 and the CheW-binding P5 domains is bound by two limits differing by ∼20°. In one limit, Tm14 aligns roughly along P3 and may interact to some extent with the hinge region near the P3 hairpin loop. In the other limit, Tm14 tilts to interact with the P5 domain of the opposite subunit in an interface that mimics that observed with the P5 homologue CheW. The time domain ESR data can be simulated from the model only if orientational variability is introduced for the P5 and, especially, P3 domains. The Tm14 tip also binds beside one of the CheA kinase domains (P4); however, in both bound and unbound states, P4 samples a broad range of distributions that are only minimally affected by Tm14 binding. The CheA P1 domains that contain the substrate histidine are also broadly distributed in space under all conditions. In the context of the hexagonal lattice formed by trimeric transmembrane chemoreceptors, the PDS structure is best accommodated with the P3 domain in the center of a honeycomb edge.

The mammalian circadian clock synchronizes physical and metabolic activity with the diurnal cycle through a transcriptional−posttranslational feedback loop. An additional feedback mechanism regulating clock timing has been proposed to involve oscillation in heme availability. Period 2 (PER2), an integral component in the negative feedback loop that establishes circadian rhythms in mammals, has been identified as a heme-binding protein. However, the majority of evidence for heme binding is based upon in vitro heme-binding assays. We sought to ascertain if these largely spectral assays could distinguish between specific and nonspecific heme interactions. Heme-binding properties by a number of other well-characterized proteins, all with no known biological role involving heme interaction, corresponded to those displayed by PER2. Site-directed mutants of putative heme-binding residues identified by MCD were unable to locate a specific heme-binding site on PER2. Protein film electrochemistry also indicates that heme binds PER2 nonspecifically on the protein surface. Our results establish the inability of qualitative in vitro assays to easily distinguish between specific and nonspecific heme binding. We conclude that heme binding to PER2 is likely to be nonspecific and does not involve the hydrophobic pocket within the PER2 PAS domains that in other PAS proteins commonly recognizes cofactors. These findings also question the significance of in vivo studies that implicate heme interactions with the clock proteins PER2 and nPAS2 in biological function.

Photoinduced relaxation processes of five structurally characterized Pseudomonas aeruginosa ReI(CO)3(α-diimine)(HisX) (X = 83, 107, 109, 124, 126)CuII azurins have been investigated by time-resolved (ps−ns) IR spectroscopy and emission spectroscopy. Crystal structures reveal the presence of Re-azurin dimers and trimers that in two cases (X = 107, 124) involve van der Waals interactions between interdigitated diimine aromatic rings. Time-dependent emission anisotropy measurements confirm that the proteins aggregate in mM solutions (D2O, KPi buffer, pD = 7.1). Excited-state DFT calculations show that extensive charge redistribution in the ReI(CO)3 → diimine 3MLCT state occurs: excitation of this 3MLCT state triggers several relaxation processes in Re-azurins whose kinetics strongly depend on the location of the metallolabel on the protein surface. Relaxation is manifested by dynamic blue shifts of excited-state ν(CO) IR bands that occur with triexponential kinetics: intramolecular vibrational redistribution together with vibrational and solvent relaxation give rise to subps, ∼2, and 8−20 ps components, while the ∼102 ps kinetics are attributed to displacement (reorientation) of the ReI(CO)3(phen)(im) unit relative to the peptide chain, which optimizes Coulombic interactions of the ReI excited-state electron density with solvated peptide groups. Evidence also suggests that additional segmental movements of Re-bearing β-strands occur without perturbing the reaction field or interactions with the peptide. Our work demonstrates that time-resolved IR spectroscopy and emission anisotropy of ReI carbonyl−diimine complexes are powerful probes of molecular dynamics at or around the surfaces of proteins and protein−protein interfacial regions.

Cryoreduction EPR/ENDOR/step-annealing measurements with substrate complexes of oxy-gsNOS (3; gsNOS is nitric oxide synthase from Geobacillus stearothermophilus) confirm that Compound I (6) is the reactive heme species that carries out the gsNOS-catalyzed (Stage I) oxidation of l-arginine to N-hydroxy-l-arginine (NOHA), whereas the active species in the (Stage II) oxidation of NOHA to citrulline and HNO/NO− is the hydroperoxy-ferric form (5). When 3 is reduced by tetrahydrobiopterin (BH4), instead of an externally supplied electron, the resulting BH4+ radical oxidizes HNO/NO− to NO. In this report, radiolytic one-electron reduction of 3 and its complexes with Arg, Me-Arg, and NO2Arg was shown by EPR and 1H and 14,15N ENDOR spectroscopies to generate 5; in contrast, during cryoreduction of 3/NOHA, the peroxo-ferric-gsNOS intermediate (4/NOHA) was trapped. During annealing at 145 K, ENDOR shows that 5/Arg and 5/Me-Arg (but not 5/NO2Arg) generate a Stage I primary product species in which the OH group of the hydroxylated substrate is coordinated to Fe(III), characteristic of 6 as the active heme center. Analysis shows that hydroxylation of Arg and Me-Arg is quantitative. Annealing of 4/NOHA at 160 K converts it first to 5/NOHA and then to the Stage II primary enzymatic product. The latter contains Fe(III) coordinated by water, characteristic of 5 as the active heme center. It further contains quantitative amounts of citrulline and HNO/NO−; the latter reacts with the ferriheme to form the NO-ferroheme upon further annealing. Stage I delivery of the first proton of catalysis to the (unobserved) 4 formed by cryoreduction of 3 involves a bound water that may convey a proton from l-Arg, while the second proton likely derives from the carboxyl side chain of Glu 248 or the heme carboxylates; the process also involves proton delivery by water(s). In the Stage II oxidation of NOHA, the proton that converts 4/NOHA to 5/NOHA likely is derived from NOHA itself, a conclusion supported by the pH invariance of the process. The present results illustrate how the substrate itself modulates the nature and reactivity of intermediates along the monooxygenase reaction pathway.

Transmembrane chemoreceptors, also known as methyl-accepting chemotaxis proteins (MCPs), translate extracellular signals into intracellular responses in the bacterial chemotaxis system. MCP ligand binding domains control the activity of the CheA kinase, situated ∼200 Å away, across the cytoplasmic membrane. The 2.17 Å resolution crystal structure of a Thermotoga maritima soluble receptor (Tm14) reveals distortions in its dimeric four-helix bundle that provide insight into the conformational states available to MCPs for propagating signals. A bulge in one helix generates asymmetry between subunits that displaces the kinase-interacting tip, which resides more than 100 Å away. The maximum bundle distortion maps to the adaptation region of transmembrane MCPs where reversible methylation of acidic residues tunes receptor activity. Minor alterations in coiled-coil packing geometry translate the bulge distortion to a >25 Å movement of the tip relative to the bundle stalks. The Tm14 structure discloses how alterations in local helical structure, which could be induced by changes in methylation state and/or by conformational signals from membrane proximal regions, can reposition a remote domain that interacts with the CheA kinase.

Deinococcus radiodurans (Dr) withstands desiccation, reactive oxygen species, and doses of radiation that would be lethal to most organisms. Deletion of a gene encoding a homolog of mammalian nitric oxide synthase (NOS) severely compromises the recovery of Dr from ultraviolet (UV) radiation damage. The Δnos defect can be complemented with recombinant NOS, rescued by exogenous nitric oxide (NO) and mimicked in the wild-type strain with an NO scavenging compound. UV radiation induces both upregulation of the nos gene and cellular NO production on similar time scales. Growth recovery does not depend on NO being present during UV irradiation, but rather can be manifested by NO addition hours after exposure. Surprisingly, nos deletion does not increase sensitivity to oxidative damage, and hydrogen peroxide does not induce nos expression. However, NOS-derived NO upregulates transcription of obgE, a gene involved in bacterial growth proliferation and stress response. Overexpression of the ObgE GTPase in the Δnos background substantially alleviates the growth defect after radiation damage. Thus, NO acts as a signal for the transcriptional regulation of growth in D. radiodurans.

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal β-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap−PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV−LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

Abstract

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).

Although bonding networks determine electron-transfer (ET) rates within proteins, the mechanism by which structure and dynamics influence ET across protein interfaces is not well understood. Measurements of photochemically induced ET and subsequent charge recombination between Zn-porphyrin-substituted cytochrome c peroxidase and cytochrome c in single crystals correlate reactivity with defined structures for different association modes of the redox partners. Structures and ET rates in crystals are consistent with tryptophan oxidation mediating charge recombination reactions. Conservative mutations at the interface can drastically affect how the proteins orient and dispose redox centers. Whereas some configurations are ET inactive, the wild-type complex exhibits the fastest recombination rate. Other association modes generate ET rates that do not correlate with predictions based on cofactor separations or simple bonding pathways. Inhibition of photoinduced ET at <273 K indicates gating by small-amplitude dynamics, even within the crystal. Thus, different associations achieve states of similar reactivity, and within those states conformational fluctuations enable interprotein ET.

In mammals, nitric oxide synthases (NOSs) produce nitric oxide for signaling and defense functions; in Streptomyces, NOS proteins nitrate a tryptophanyl moiety in synthesis of a phytotoxin. We have discovered that the NOS protein from the radiation-resistant bacterium Deinococcus radiodurans (deiNOS) associates with an unusual tryptophanyl tRNA synthetase (TrpRS). D. radiodurans contains genes for two TrpRSs: the first has ≈40% sequence identity to typical TrpRSs, whereas the second, identified as the NOS-interacting protein (TrpRS II), has only ≈29% identity. TrpRS II is induced after radiation damage and contains an N-terminal extension similar to those of proteins involved in stress responses. Recombinantly expressed TrpRS II binds tryptophan (Trp), ATP, and D. radiodurans tRNATrp and catalyzes the formation of 5′ adenyl-Trp and tRNATrp, with approximately five times less activity than TrpRS I. Upon coexpression in Escherichia coli, TrpRS II binds to, copurifies with, and dramatically enhances the solubility of deiNOS. Dimeric TrpRS II binds dimeric deiNOS with a stoichiometry of 1:1 and a dissociation constant of 6–30 μM. Upon forming a complex, deiNOS quenches the fluorescence of an ATP analog bound to TrpRS II, and increases its affinity for substrate l-arginine. Remarkably, TrpRS II also activates the NOS activity of deiNOS. These findings reveal a link between bacterial NOS and Trp metabolism in a second organism and may indicate yet another novel biological function for bacterial NOS.

Cytochromes P450 play key roles in drug metabolism and disease by oxidizing a wide variety of natural and xenobiotic compounds. High-resolution crystal structures of P450cam bound to ruthenium sensitizer-linked substrates reveal an open conformation of the enzyme that allows substrates to access the active center via a 22-Å deep channel. Interactions of alkyl and fluorinated biphenyl linkers with the channel demonstrate the importance of exploiting protein dynamics for specific inhibitor design. Large changes in peripheral enzyme structure (F and G helices) couple to conformational changes in active center residues (I helix) implicated in proton pumping and dioxygen activation. Common conformational states among P450cam and homologous enzymes indicate that static and dynamic variability in the F/G helix region allows the 54 human P450s to oxidize thousands of substrates.

Over-expression of heme binding proteins in Escherichia coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in E. coli often contain less than a full complement of heme because they rather are partially incorporated with free-base porphyrin. Porphyrin-incorporated proteins have similar spectral characteristics as the desired heme-loaded targets, and thus are difficult to detect, even in purified samples. We present a straightforward and inexpensive solution to this problem that involves the co-expression of native ferrochelatase with the protein of interest. The method is shown to be effective for proteins that contain either Cys- or His-ligated hemes.

Nitric oxide synthases (NOSs) are heme-based monooxygenases that oxidize L-arginine to nitric oxide (NO), a signaling molecule and cytotoxic agent in higher organisms. Although NOS-like activity has been reported in many bacteria, only a few bacterial homologs of mammalian NOSs (mNOSs) have been characterized to date. In contrast to mNOSs, which possess both a catalytic and a reductase domain, the bacterial enzymes lack reductase domains and require the supply of suitable reductants to produce NO. A notable exception is a NOS from a gram-negative bacterium that contains a new type of reductase module. Remarkably, bacterial NOSs seem to have functions that differ from those of mNOSs, including nitration of different metabolites and protection against oxidative stress. Studies of bacterial NOSs will probably result in a better understanding of the mechanism of NO synthesis and unveil a variety of new functions for NO in microbes.

We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to report on the conformation of Cu-Zn superoxide dismutase (SOD1) through the sensitive measurement of dipolar interactions between inherent Cu2+ ions. Although the extent and the anisotropy of the Cu ESR spectrum provides challenges for PDS, Ku-band (17.3 GHz) double electron-electron resonance and double-quantum coherence variants of PDS coupled with distance reconstruction methods recover Cu-Cu distances in good agreement with crystal structures. Moreover, Cu-PDS measurements expose distinct differences between the conformational properties of wild-type SOD1 and a single-residue variant (I149T) that leads to the disease amyotrophic lateral sclerosis (ALS). The I149T protein displays a broader Cu-Cu distance distribution within the SOD1 dimer compared to wild-type. In a nitroxide (NO)-labeled sample, distance distributions obtained from Cu-Cu, Cu-NO, and NO-NO separations reveal increased structural heterogeneity within the protein and a tendency for mutant dimers to associate. In contrast, perturbations caused by the ALS mutation are completely masked in the crystal structure of I149T. Thus, PDS readily detects alterations in metalloenzyme solution properties not easily deciphered by other methods and in doing so supports the notion that increased range of motion and associations of SOD1 ALS variants contribute to disease progression.

•FliFC:FliGN fold together to produce a topology that repeats throughout FliG•FliFC:FliGN interact through hydrophobic contacts critical for motor function•FliF:FliG 1:1 stoichiometry produces an MS/C-ring interface with ∼C25-fold symmetryThe interface between the membrane (MS) and cytoplasmic (C) rings of the bacterial flagellar motor couples torque generation to rotation within the membrane. The structure of the C-terminal helices of the integral membrane protein FliF (FliFC) bound to the N terminal domain of the switch complex protein FliG (FliGN) reveals that FliGN folds around FliFC to produce a topology that closely resembles both the middle and C-terminal domains of FliG. The interface is consistent with solution-state nuclear magnetic resonance, small-angle X-ray scattering, in vivo interaction studies, and cellular motility assays. Co-folding with FliFC induces substantial conformational changes in FliGN and suggests that FliF and FliG have the same stoichiometry within the rotor. Modeling the FliFC:FliGN complex into cryo-electron microscopy rotor density updates the architecture of the middle and upper switch complex and shows how domain shuffling of a conserved interaction module anchors the cytoplasmic rotor to the membrane.Download high-res image (244KB)Download full-size image

The PAS–LOV domain is a signal-transducing component found in a large variety of proteins that is responsible for sensing different stimuli such as light, oxygen, and voltage. The LOV protein VVD regulates blue light responses in the filamentous fungi Neurospora crassa. Using photocoupled, time-resolved small-angle X-ray scattering, we extract the solution protein structure in both dark-adapted and light-activated states. Two distinct dark-adapted conformations are detected in the wild-type protein: a compact structure that corresponds to the crystal structure of the dark-state monomer as well as an extended structure that is well modeled by introducing conformational disorder at the N-terminus of the protein. These conformations are accentuated in carefully selected variants, in which a key residue for propagating structural transitions, Cys71, has been mutated or oxidized. Despite different dark-state conformations, all proteins form a common dimer in response to illumination. Taken together, these data support a reaction scheme that describes the mechanism for light-induced dimerization of VVD. Envelope reconstructions of the transient light-state dimer reveal structures that are best described by a parallel arrangement of subunits that have significantly changed conformation compared to the crystal structure.

•The chemotaxis histidine kinase CheA has on-state and off-state that depend on conformational dynamics.•The substrate domain (P1) is sequestered in the off-state by interactions that involve the adjacent subunit.•Structural changes at the ATP pocket relieve P1 inhibition.•The structure of the minimal active unit containing the kinase and dimerization domain is consistent with receptor array integration and explains trans subunit phosphorylation.During bacterial chemotaxis, transmembrane chemoreceptor arrays regulate autophosphorylation of the dimeric histidine kinase CheA. The five domains of CheA (P1–P5) each play a specific role in coupling receptor stimulation to CheA activity. Biochemical and X-ray scattering studies of thermostable CheA from Thermotoga maritima determine that the His-containing substrate domain (P1) is sequestered by interactions that depend upon P1 of the adjacent subunit. Non-hydrolyzable ATP analogs (but not ATP or ADP) release P1 from the protein core (domains P3P4P5) and increase its mobility. Detachment of both P1 domains or removal of one within a dimer increases net autophosphorylation substantially at physiological temperature (55 °C). However, nearly all activity is lost without the dimerization domain (P3). The linker length between P1 and P3 dictates intersubunit (trans) versus intrasubunit (cis) autophosphorylation, with the trans reaction requiring a minimum length of 47 residues. A new crystal structure of the most active dimerization-plus-kinase unit (P3P4) reveals trans directing interactions between the tether connecting P3 to P2–P1 and the adjacent ATP-binding (P4) domain. The orientation of P4 relative to P3 in the P3P4 structure supports a planar CheA conformation that is required by membrane array models, and it suggests that the ATP lid of CheA may be poised to interact with receptors and coupling proteins. Collectively, these data suggest that the P1 domains are restrained in the off-state as a result of cross-subunit interactions. Perturbations at the nucleotide-binding pocket increase P1 mobility and access of the substrate His to P4-bound ATP.Download high-res image (256KB)Download full-size image

Period (PER) is the major transcription inhibitor in metazoan circadian clocks and lies at the center of several feedback loops that regulate gene expression. Dimerization of Drosophila PER influences nuclear translocation, repressor activity, and behavioral rhythms. The structure of a central, 346-residue PER fragment reveals two associated PAS (Per-Arnt-Sim) domains followed by a protruding α-helical extension (αF). A closed, pseudo-symmetric dimer forms from a cross handshake interaction of the N-terminal PAS domain with αF of the opposing subunit. Strikingly, a shift of αF against the PAS β-sheet generates two alternative subunit interfaces in the dimer. Taken together with a previously reported PER structure in which αF extends, these data indicate that αF unlatches to switch association of PER with itself to its partner Timeless. The variable positions of the αF helix provide snapshots of a helix dissociation mechanism that has relevance to other PAS protein systems. Conservation of PER interaction residues among a family of PAS-AB-containing transcription factors suggests that contacts mediating closed PAS-AB dimers serve a general function.Download high-res image (166KB)Download full-size imageHighlights► PER dimerization influences translocation, activity, and behavioral rhythms. ► The structure of a central PER fragment reveals two associated PAS domains. ► A closed, pseudo-symmetric dimer is formed by the PER PAS domain homology region. ► A helical extension that follows the PAS domain latches the dimer together. ► This helical extension can assume multiple positions.

Biologically important protein complexes often involve molecular interactions that are low affinity or transient. We apply pulsed dipolar electron spin resonance spectroscopy and site-directed spin labeling in what to our knowledge is a new approach to study aggregation and to identify regions on protein surfaces that participate in weak, but specific molecular interactions. As a test case, we have probed the self-association of the chemotaxis kinase CheA, which forms signaling clusters with chemoreceptors and the coupling protein CheW at the poles of bacterial cells. By measuring the intermolecular dipolar interactions sensed by spin-labels distributed over the protein surface, we show that the soluble CheA kinase aggregates to a small extent through interactions mediated by its regulatory (P5) domain. Direct dipolar distance measurements confirm that a hydrophobic surface at the periphery of P5 subdomain 2 associates CheA dimers in solution. This result is further supported by differential disulfide cross-linking from engineered cysteine reporter sites. We suggest that the periphery of P5 is an interaction site on CheA for other similar hydrophobic surfaces and plays an important role in structuring the signaling particle.

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 three-unit poly-HAMP chain from the Pseudomonas 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 angle, 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.Highlights► Presents the first poly-HAMP structure and identifies a novel HAMP domain conformation ► HAMP conformations differ by changes in helical register, rotation, and crossing angle ► Proposes a new signal transduction model consistent with known signal inputs to HAMP ► Provides an output mechanism for HAMP domains involving stutter compensation

Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here, we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS (Per–Arnt–Sim) and poly-HAMP (histidine kinase–adenylyl cyclase–methyl-accepting chemotaxis protein–phosphatase) domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state, identifies conformational changes induced by ligand binding that are likely essential for the signaling mechanism. Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS but propagate to the Iβ strand in a manner predicted to alter PAS–PAS associations and the downstream HAMP junction within full-length Aer2. Small-angle X-ray scattering of PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initio small-angle X-ray scattering molecular envelopes, and pulsed dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the Escherichia coli Aer paradigm, where side-on PAS–HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains.Download high-res image (398KB)Download full-size imageHighlights► We demonstrate that similar PAS domains can display distinctly different heme-binding modes and ligand-sensing mechanisms. ► This study reveals that ligand binding induces a global structural rearrangement in the Aer2 PAS domain. ► We propose an in-line model for PAS–HAMP signaling that differs from the E. coli Aer paradigm.

Re-investigation of the electrochemical behavior of the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin on graphite electrodes has revealed drastic differences in reversibility of electron transfer (ET) depending on the type of electrode surface employed. In particular, slow electron transfer kinetics and quasireversibility on an unpolished glassy carbon electrode can mask underlying concerted two-electron transfer chemistry and cause the appearance of an apparent one-electron couple. Nonetheless, the thermodynamic instability of the radical intermediate prevents any detectable build-up of this intermediate under any conditions tested. Scan rate and pH-dependencies of the concerted two-electron couple indicate a kinetic barrier to formation of the radical that depends on proton availability. These observations resolve previous conflicting interpretations of tetrahydrobiopterin solution electrochemistry and comment on how NOS may stabilize the one-electron oxidized radical state that participates in enzymatic production of nitric oxide.