Structural and chemical requirements for histidine phosphorylation by the chemotaxis kinase CheA

The CheA histidine kinase initiates the signal transduction pathway of bacterial chemotaxis by autophosphorylating a conserved histidine on its phosphotransferase domain (P1). Site-directed mutations of neighboring conserved P1 residues (Glu-67, Lys-48, and His-64) show that a hydrogen-bonding network controls the reactivity of the phospho-accepting His (His-45) in Thermotoga maritima CheA. In particular, the conservative mutation E67Q dramatically reduces phospho-transfer to P1 without significantly affecting the affinity of P1 for the CheA ATP-binding domain. High resolution crystallographic studies revealed that although all mutants disrupt the hydrogen-bonding network to varying degrees, none affect the conformation of His-45. N-15-NMR chemical shift studies instead showed that Glu-67 functions to stabilize the unfavored (NH)-H-delta 1 tautomer of His-45, thereby rendering the N-epsilon 2 imidazole unprotonated and well positioned for accepting the ATP phosphoryl group

Electron Tunneling in Single Crystals of Pseudomonas aeruginosa Azurins

Rates of reduction of Os(III), Ru(III), and Re(I)^* by Cu(I) in His83-modified Pseudomonas aeruginosa azurins (M−Cu distance ∼17 Å) have been measured in single crystals, where protein conformation and surface solvation are precisely defined by high-resolution X-ray structure determinations:  1.7(8) × 10^6 s^(-1) (298 K), 1.8(8) × 10^6 s^(-1) (140 K), [Ru(bpy)_2(im)^(3+)-]; 3.0(15) × 10^6 s^(-1) (298 K), [Ru(tpy)(bpy)^(3+)-]; 3.0(15) × 10^6 s^(-1) (298 K), [Ru(tpy)(phen)^(3+)-]; 9.0(50) × 10^2 s^(-1) (298 K), [Os(bpy)2(im)^(3+)-]; 4.4(20) × 10^6 s^(-1) (298 K), [Re(CO)_3(phen)^(+*)] (bpy = 2,2‘-bipyridine; im = imidazole; tpy = 2,2‘:6‘,2‘ ‘-terpyridine; phen = 1,10-phenanthroline). The time constants for electron tunneling in crystals are roughly the same as those measured in solution, indicating very similar protein structures in the two states. High-resolution structures of the oxidized (1.5 Å) and reduced (1.4 Å) states of Ru(II)(tpy)(phen)(His83)Az establish that very small changes in copper coordination accompany reduction but reveal a shorter axial interaction between copper and the Gly45 peptide carbonyl oxygen [2.6 Å for Cu(II)] than had been recognized previously. Although Ru(bpy)_2(im)(His83)Az is less solvated in the crystal, the reorganization energy for Cu(I) → Ru(III) electron transfer falls in the range (0.6−0.8 eV) determined experimentally for the reaction in solution. Our work suggests that outer-sphere protein reorganization is the dominant activation component required for electron tunneling

Structure of an Enclosed Dimer Formed by the Drosophila Period Protein

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, pseudosymmetric 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

Relaxation Dynamics of Pseudomonas aeruginosa Re^I(C)O_3(α-diimine)(HisX)^+ (X=83, 107, 109, 124, 126)Cu-^(II) Azurins

Photoinduced relaxation processes of five structurally characterized Pseudomonas aeruginosa Re^I(CO)_3(α-diimine)(HisX) (X = 83, 107, 109, 124, 126)Cu^(II) 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 ~10^2 ps kinetics are attributed to displacement (reorientation) of the Re^I(CO)_3(phen)(im) unit relative to the peptide chain, which optimizes Coulombic interactions of the Re^I 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 Re^I carbonyl−diimine complexes are powerful probes of molecular dynamics at or around the surfaces of proteins and protein−protein interfacial regions

Atypical chemoreceptor arrays accommodate high membrane curvature

The prokaryotic chemotaxis system is arguably the best-understood signaling pathway in biology. In all previously described species, chemoreceptors organize into a hexagonal (P6 symmetry) extended array. Here, we report an alternative symmetry (P2) of the chemotaxis apparatus that emerges from a strict linear organization of the histidine kinase CheA in Treponema denticola cells, which possesses arrays with the highest native curvature investigated thus far. Using cryo-ET, we reveal that Td chemoreceptor arrays assume an unusual arrangement of the supra-molecular protein assembly that has likely evolved to accommodate the high membrane curvature. The arrays have several atypical features, such as an extended dimerization domain of CheA and a variant CheW-CheR-like fusion protein that is critical for maintaining an ordered chemosensory apparatus. Furthermore, the previously characterized Td oxygen sensor ODP influences CheA ordering. These results suggest a greater diversity of the chemotaxis signaling system than previously thought

Spectroscopy and Reactivity of a Photogenerated Tryptophan Radical in a Structurally Defined Protein Environment

Near-UV irradiation of structurally characterized [Re(I)(CO)_3(1,10-phenanthroline)(Q107H)](W48F/Y72F/H83Q/Y108W)AzM(II) [Az = Pseudomonas aeruginosa azurin, M = Cu, Zn]/[Co(NH_3)_5Cl]Cl_2 produces a tryptophan radical (W108•) with unprecedented kinetic stability. After rapid formation (k = 2.8 × 10^6 s^(-1)), the radical persists for more than 5 h at room temperature in the folded ReAzM(II) structure. The absorption spectrum of ReAz(W108•)M(II) exhibits maxima at 512 and 536 nm. Oxidation of K_4[Mo(CN)_8] by ReAz(W108•)Zn(II) places the W108•/W108 reduction potential in the protein above 0.8 V vs NHE

Signal transduction in light-oxygen-voltage receptors lacking the adduct- forming cysteine residue

Light–oxygen–voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct- forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications

Structures of Ruthenium-modified Pseudomonas aeruginosa Azurin and [Ru(2,2’-bipyridine)_2(imidazole)_2)]SO_4•10H_2O

The crystal structure of Ru(2,2'-bipyridine)_2(imidazole)(His83)azurin (RuAz) has been determined to 2.3 Å ¬resolution by X-ray crystallography. The spectroscopic and thermodynamic properties of both the native protein and [Ru(2,2'-bipyridine)_2(imidazole)_2]^(2+) are maintained in the modified protein. Dark-green RuAz crystals grown from PEG 4000, LiNO_3, CuCl_2 and Tris buffer are monoclinic, belong to the space group C2 and have cell parameters a = 100.6, b = 35.4, c = 74.7 Å and β = 106.5°. In addition, [Ru(2,2'-bipyridine)_2(imidazole)_2]SO_4•10H_2O was synthesized, crystallized and structurally characterized by X-ray crystallography. Red-brown crystals of this complex are monoclinic, space group P2_1/n, unit-cell parameters a = 13.230 (2), b = 18.197 (4), c = 16.126 (4) Å, β = 108.65 (2)°. Stereochemical parameters for the refinement of Ru(2,2'-bipyridine)_2(imidazole)(His83) were taken from the atomic coordinates of [Ru(2,2'-bipyridine)_2(imidazole)_2]^(2+). The structure of RuAz confirms that His83 is the only site of chemical modification and that the native azurin structure is not perturbed significantly by the ruthenium label

Bacterial chemoreceptor arrays are hexagonally packed trimers of receptor dimers networked by rings of kinase and coupling proteins

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