Mark J. Banfield

A divergent external loop confers antagonistic activity on floral regulators FT and TFL1

The Arabidopsis genes FT and TERMINAL FLOWER1 (TFL1) encode related proteins with similarity to human Raf kinase inhibitor protein. FT, and likely also TFL1, is recruited to the promoters of floral genes through interaction with FD, a bZIP transcription factor. FT, however, induces flowering, while TFL1 represses flowering. Residues responsible for the opposite activities of FT and TFL1 were mapped by examining plants that overexpress chimeric proteins. A region important in vivo localizes to a 14‐amino‐acid segment that evolves very rapidly in TFL1 orthologs, but is almost invariant in FT orthologs. Crystal structures show that this segment forms an external loop of variable conformation. The only residue unambiguously distinguishing the FT and TFL1 loops makes a hydrogen bond with a residue near the entrance of a potential ligand‐binding pocket in TFL1, but not in FT. This pocket is contacted by a C‐terminal peptide, which also contributes to the opposite FT and TFL1 activities. In combination, these results identify a molecular surface likely to be recognized by FT‐ and/or TFL1‐specific interactors.

Protein-Folding Location Can Regulate Manganese-Binding Versus Copper- or Zinc-Binding.

Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving–Williams series, Cu2+ and Zn2+ typically form more stable complexes than Mn2+. Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu2+-protein, CucA (Cu2+-cupin A), and the most abundant Mn2+-protein, MncA (Mn2+-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving–Williams series, MncA only binds Mn2+ after folding in solutions containing at least a 104 times molar excess of Mn2+ over Cu2+ or Zn2+. However once MncA has bound Mn2+, the metal does not exchange with Cu2+. MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn2+ (refs 10–12) but micromolar Mn2+ (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu2+. These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn2+.

Oomycetes, effectors, and all that jazz

Plant pathogenic oomycetes secrete a diverse repertoire of effector proteins that modulate host innate immunity and enable parasitic infection. Understanding how effectors evolve, translocate and traffic inside host cells, and perturb host processes are major themes in the study of oomycete-plant interactions. The last year has seen important progress in the study of oomycete effectors with, notably, the elucidation of the 3D structures of five RXLR effectors, and novel insights into how cytoplasmic effectors subvert host cells. In this review, we discuss these and other recent advances and highlight the most important open questions in oomycete effector biology.

DETERMINATE and LATE FLOWERING Are Two TERMINAL FLOWER1/CENTRORADIALIS Homologs That Control Two Distinct Phases of Flowering Initiation and Development in Pea

Genes in the TERMINAL FLOWER1 (TFL1)/CENTRORADIALIS family are important key regulatory genes involved in the control of flowering time and floral architecture in several different plant species. To understand the functions of TFL1 homologs in pea, we isolated three TFL1 homologs, which we have designated PsTFL1a, PsTFL1b, and PsTFL1c. By genetic mapping and sequencing of mutant alleles, we demonstrate that PsTFL1a corresponds to the DETERMINATE (DET) gene and PsTFL1c corresponds to the LATE FLOWERING (LF) gene. DET acts to maintain the indeterminacy of the apical meristem during flowering, and consistent with this role, DET expression is limited to the shoot apex after floral initiation. LF delays the induction of flowering by lengthening the vegetative phase, and allelic variation at the LF locus is an important component of natural variation for flowering time in pea. The most severe class of alleles flowers early and carries either a deletion of the entire PsTFL1c gene or an amino acid substitution. Other natural and induced alleles for LF, with an intermediate flowering time phenotype, present no changes in the PsTFL1c amino acid sequence but affect LF transcript level in the shoot apex: low LF transcript levels are correlated with early flowering, and high LF transcript levels are correlated with late flowering. Thus, different TFL1 homologs control two distinct aspects of plant development in pea, whereas a single gene, TFL1, performs both functions in Arabidopsis. These results show that different species have evolved different strategies to control key developmental transitions and also that the genetic basis for natural variation in flowering time may differ among plant species.

Function from structure? The crystal structure of human phosphatidylethanolamine-binding protein suggests a role in membrane signal transduction.

BACKGROUND Proteins belonging to the phosphatidylethanolamine-binding protein (PEBP) family are highly conserved throughout nature and have no significant sequence homology with other proteins of known structure or function. A variety of biological roles have previously been described for members of this family, including lipid binding, roles as odorant effector molecules or opioids, interaction with the cell-signalling machinery, regulation of flowering plant stem architecture, and a function as a precursor protein of a bioactive brain neuropeptide. To date, no experimentally derived structural information has been available for this protein family. In this study we have used X-ray crystallography to determine the three-dimensional structure of human PEBP (hPEBP), in an attempt to clarify the biological role of this unique protein family. RESULTS The crystal structures of two forms of hPEBP have been determined: one in the native state (at 2.05 A resolution) and one in complex with cacodylate (at 1.75 A resolution). The crystal structures reveal that hPEBP adopts a novel protein topology, dominated by the presence of a large central beta sheet, and is expected to represent the archaetypal fold for this family of proteins. Two potential functional sites have been identified from the structure: a putative ligand-binding site and a coupled cleavage site. hPEBP forms a dimer in the crystal with a distinctive dipole moment that may orient the oligomer for membrane binding. CONCLUSIONS The crystal structure of hPEBP suggests that the ligand-binding site could accommodate the phosphate head groups of membrane lipids, therefore allowing the protein to adhere to the inner leaf of bilipid membranes where it would be ideally positioned to relay signals from the membrane to the cytoplasm. The structure also suggests that ligand binding may lead to coordinated release of the N-terminal region of the protein to form the hippocampal neurostimulatory peptide, which is known to be active in the development of the hippocampus. These studies are consistent with a primary biological role for hPEBP as a transducer of signals from the interior membrane surface.

Structures of Phytophthora RXLR effector proteins: a conserved but adaptable fold underpins functional diversity.

Phytopathogens deliver effector proteins inside host plant cells to promote infection. These proteins can also be sensed by the plant immune system, leading to restriction of pathogen growth. Effector genes can display signatures of positive selection and rapid evolution, presumably a consequence of their co-evolutionary arms race with plants. The molecular mechanisms underlying how effectors evolve to gain new virulence functions and/or evade the plant immune system are poorly understood. Here, we report the crystal structures of the effector domains from two oomycete RXLR proteins, Phytophthora capsici AVR3a11 and Phytophthora infestans PexRD2. Despite sharing <20% sequence identity in their effector domains, they display a conserved core α-helical fold. Bioinformatic analyses suggest that the core fold occurs in ∼44% of annotated Phytophthora RXLR effectors, both as a single domain and in tandem repeats of up to 11 units. Functionally important and polymorphic residues map to the surface of the structures, and PexRD2, but not AVR3a11, oligomerizes in planta. We conclude that the core α-helical fold enables functional adaptation of these fast evolving effectors through (i) insertion/deletions in loop regions between α-helices, (ii) extensions to the N and C termini, (iii) amino acid replacements in surface residues, (iv) tandem domain duplications, and (v) oligomerization. We hypothesize that the molecular stability provided by this core fold, combined with considerable potential for plasticity, underlies the evolution of effectors that maintain their virulence activities while evading recognition by the plant immune system.

Crystal Structure of Streptococcus pyogenes Sortase A: Implications for Sortase mechanism

Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a “housekeeping” sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design.

Recent developments in effector biology of filamentous plant pathogens

Ricardo Oliva,1 Joe Win,1 Sylvain Raffaele,1 Laurence Boutemy,2 Tolga O. Bozkurt,1 Angela Chaparro-Garcia,1 Maria Eugenia Segretin,1 Remco Stam,1 Sebastian Schornack,1 Liliana M. Cano,1 Mireille van Damme,1 Edgar Huitema,3 Marco Thines,1,4 Mark J. Banfield2 and Sophien Kamoun1* The Sainsbury Laboratory, Norwich NR4 7UH, UK. Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, UK. Division of Plant Sciences, College of Life Sciences, University of Dundee at Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK. University of Hohenheim, Institute of Botany 210, 70593 Stuttgart, Germany.

Direct observation of the iron binding sites in a ferritin

X‐Ray analysis of the ferritin of Escherichia coli (Ec‐FTN) and of Ec‐FTN crystals soaked in (NH4)2Fe(SO4)2 has revealed the presence of three iron‐binding sites per subunit. Two of these form a di‐iron site in the centre of the subunit as has been proposed for the ‘ferroxidase centres’ of human ferritin H chains. This di‐iron site, lying within the 4‐alpha‐helix bundle, resemble those of ribonucleotide reductase, methane monoxygenase and haemerythrin. The third iron is bound by ligands unique to Ec‐FTN on the inner surface of the protein shell. It is speculated that this state may represent the nucleation centre of a novel type of Fe(III) cluster, recently observed in Ec‐FTN.

Sequence Divergent RXLR Effectors Share a Structural Fold Conserved across Plant Pathogenic Oomycete Species

The availability of genome sequences for some of the most devastating eukaryotic plant pathogens has led a revolution in our understanding of how these parasites cause disease, and how their hosts respond to invasion [1]–[7]. One of the most significant discoveries from the genome sequences of plant pathogenic oomycetes is the plethora of putative translocated effector proteins these organisms encode. Many effector genes display signatures of rapid evolution and tend to reside in dynamic regions of the pathogen genomes. Once inside the host, effector proteins modulate cellular processes, mainly suppressing plant immunity [8]–[12]. Effectors can also be recognized directly or indirectly by the plant immune system through the action of disease resistance (R) proteins [13], [14].