James W. Murray

X-ray crystallography identifies two chloride binding sites in the oxygen evolving centre of Photosystem II

Bromide anomalous X-ray diffraction analyses have been used to locate chloride binding sites in the vicinity of the water splitting/oxygen evolving centre (OEC) of Photosystem II. Three-dimensional crystals of PSII from Thermosynechococcus elongatus were grown from (i) isolated PSII crystals infiltrated with bromide or (ii) PSII obtained from cells cultured in a medium in which the chloride content was totally replaced by bromide. In either case, the anomalous diffraction yielded the same result, the existence of two bromide binding sites in the vicinity of the OEC. Neither are in the first coordination sphere of the Mn and Ca ions which form the catalytic centre of the OEC, being about 6 to 7 A from the metal-cluster. Site 1 is located close to the side chain nitrogen of D2-K317 and the backbone nitrogen of D1-Glu333 while Site 2 is adjacent to backbone nitrogens of CP43-Glu354 and D1-Asn338. Their positioning close to postulated hydrophilic channels may suggest a role in proton removal from, or substrate access to, the OEC.

Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases.

Background: BTB-Kelch proteins, including KLHL11, are proposed to bind Cul3 through a “3-box” motif to form E3 ubiquitin ligases. Results: We solved crystal structures of the KLHL11-Cul3 complex and four Kelch domains. Conclusion: The 3-box forms a hydrophobic groove that binds a specific N-terminal extension of Cul3. Significance: Dimeric BTB-Kelch proteins bind two Cul3 molecules and support a two-site model for substrate recognition. Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein “3-box” motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.

Structure and Functional Characterization of the Atypical Human Kinase Haspin.

The protein kinase haspin/Gsg2 plays an important role in mitosis, where it specifically phosphorylates Thr-3 in histone H3 (H3T3). Its protein sequence is only weakly homologous to other protein kinases and lacks the highly conserved motifs normally required for kinase activity. Here we report structures of human haspin in complex with ATP and the inhibitor iodotubercidin. These structures reveal a constitutively active kinase conformation, stabilized by haspin-specific inserts. Haspin also has a highly atypical activation segment well adapted for specific recognition of the basic histone tail. Despite the lack of a DFG motif, ATP binding to haspin is similar to that in classical kinases; however, the ATP γ-phosphate forms hydrogen bonds with the conserved catalytic loop residues Asp-649 and His-651, and a His651Ala haspin mutant is inactive, suggesting a direct role for the catalytic loop in ATP recognition. Enzyme kinetic data show that haspin phosphorylates substrate peptides through a rapid equilibrium random mechanism. A detailed analysis of histone modifications in the neighborhood of H3T3 reveals that increasing methylation at Lys-4 (H3K4) strongly decreases substrate recognition, suggesting a key role of H3K4 methylation in the regulation of haspin activity.

Parameters affecting the X-ray dose absorbed by macromolecular crystals.

The lifetime of a macromolecular crystal in an X-ray beam is assumed to be limited by the absorbed dose. This dose, expressed in Gray (Gy = J kg(-1)), is a function of a number of parameters: the absorption coefficients of the constituent atoms of the crystal, the number of molecules per asymmetric unit, the beam energy, flux, size and profile, the crystal size, and the total irradiation time. The effects of these variables on the predicted absorbed dose, calculated using the program RADDOSE, are discussed and are illustrated with reference to the irradiation of a selenomethionine protein crystal of unknown structure. The results of RADDOSE can and will in the future be used to inform the data collection procedure as it sets a theoretical upper limit on the total exposure time at a certain X-ray source. However, as illustrated with an example for which the experimental data are compared with prediction, the actual lifetime of a crystal could become shorter in those cases where specific damage breaks down crucial crystal contacts.

Colouring cryo-cooled crystals: online microspectrophotometry

A portable and readily aligned online microspectrophotometer that can be easily installed on macromolecular crystallography beamlines is described. It allows measurement of the spectral characteristics of macromolecular crystals prior, during, and after the X-ray diffraction experiment.

The structure of the Mn4Ca2+ cluster of photosystem II and its protein environment as revealed by X-ray crystallography

The location, structure and protein environment of the Mn4Ca2+ cluster, which catalyses the light-driven, water-splitting reaction of photosystem II, has been revealed by X-ray crystallography. However, owing to the low resolutions of the crystal structures reported to date, and the possibility of radiation damage at the catalytic centre, the precise position of each metal ion remains unknown. To some extent, these problems have been overcome by applying spectroscopic techniques like extended X-ray absorption fine structure. Taking into account the most recent results obtained with these two X-ray-based techniques, we have attempted to refine models of the structure of the Mn4Ca2+ cluster and its protein environment.

Structure of CyanoP at 2.8 A: implications for the evolution and function of the PsbP subunit of photosystem II .

We present here the crystal structure of CyanoP (Tlr2075) from Thermosynechococcus elongatus at 2.8 A. CyanoP is a substoichiometric component of the isolated cyanobacterial Photosystem II (PSII) complex, distantly related to the PsbP extrinsic subunit of the oxygen-evolving PSII complex in higher plants and green algae. Despite the relatively low degree of sequence similarity, we have found that CyanoP adopts the same beta-sandwich fold as higher-plant PsbP and contains a well-conserved metal (zinc)-binding site that is also present in plant PsbP. Our results support the idea that CyanoP represents the basal structural fold of the PsbP superfamily.

High-molecular-weight complexes of RsbR and paralogues in the environmental signaling pathway of Bacillus subtilis.

Bacillus subtilis has developed an intricate signal transduction cascade to respond to the imposition of a variety of stresses on the cell. Reversible protein phosphorylation and the formation of alternative protein-protein complexes modulate the activity of sigma(B), the RNA polymerase sigma factor subunit responsible for the transcription of the general stress response genes. Some of the regulators of sigma(B), such as RsbR and RsbS, are known to associate in a 25S complex, called the stressosome, that can bind RsbT until RsbT phosphorylates target residues in RsbR and RsbS. To date, the RsbR-RsbS complex appears to be the most upstream component of the sigma(B) regulatory pathway. This large structure is thought to play an important role in sensing and/or integrating signals from different physical stresses. The roles of the paralogues of RsbR that are found in B. subtilis remain unclear. We describe here how the RsbR paralogues copurify with RsbR from B. subtilis cell lysates, and we demonstrate in vitro that the paralogues form large complexes either with RsbS or with a prepurified RsbR-RsbS binary complex. We conclude from these biochemical studies that stressosomes in B. subtilis cells contain minimally RsbS and all of the RsbT-phosphorylatable RsbR paralogues.

Structural insights into serine-rich fimbriae from Gram-positive bacteria.

The serine-rich repeat family of fimbriae play important roles in the pathogenesis of streptococci and staphylococci. Despite recent attention, their finer structural details and precise adhesion mechanisms have yet to be determined. Fap1 (Fimbriae-associated protein 1) is the major structural subunit of serine-rich repeat fimbriae from Streptococcus parasanguinis and plays an essential role in fimbrial biogenesis, adhesion, and the early stages of dental plaque formation. Combining multidisciplinary, high resolution structural studies with biological assays, we provide new structural insight into adhesion by Fap1. We propose a model in which the serine-rich repeats of Fap1 subunits form an extended structure that projects the N-terminal globular domains away from the bacterial surface for adhesion to the salivary pellicle. We also uncover a novel pH-dependent conformational change that modulates adhesion and likely plays a role in survival in acidic environments.

Origin and evolution of water oxidation before the last common ancestor of the Cyanobacteria

Photosystem II, the water oxidizing enzyme, altered the course of evolution by filling the atmosphere with oxygen. Here, we reconstruct the origin and evolution of water oxidation at an unprecedented level of detail by studying the phylogeny of all D1 subunits, the main protein coordinating the water oxidizing cluster (Mn4CaO5) of Photosystem II. We show that D1 exists in several forms making well-defined clades, some of which could have evolved before the origin of water oxidation and presenting many atypical characteristics. The most ancient form is found in the genome of Gloeobacter kilaueensis JS-1 and this has a C-terminus with a higher sequence identity to D2 than to any other D1. Two other groups of early evolving D1 correspond to those expressed under prolonged far-red illumination and in darkness. These atypical D1 forms are characterized by a dramatically different Mn4CaO5 binding site and a Photosystem II containing such a site may assemble an unconventional metal cluster. The first D1 forms with a full set of ligands to the Mn4CaO5 cluster are grouped with D1 proteins expressed only under low oxygen concentrations and the latest evolving form is the dominant type of D1 found in all cyanobacteria and plastids. In addition, we show that the plastid ancestor had a D1 more similar to those in early branching Synechococcus. We suggest each one of these forms of D1 originated from transitional forms at different stages toward the innovation and optimization of water oxidation before the last common ancestor of all known cyanobacteria.