Nathan P. Cowieson

Dimerisation of a chromo shadow domain and distinctions from the chromodomain as revealed by structural analysis

BACKGROUND Proteins such as HP1, found in fruit flies and mammals, and Swi6, its fission yeast homologue, carry a chromodomain (CD) and a chromo shadow domain (CSD). These proteins are required to form functional transcriptionally silent centromeric chromatin, and their mutation leads to chromosome segregation defects. CSDs have only been found in tandem in proteins containing the related CD. Most HP1-interacting proteins have been found to associate through the CSD and many of these ligands contain a conserved pentapeptide motif. RESULTS The 1.9 A crystal structure of the Swi6 CSD is presented here. This reveals a novel dimeric structure that is distinct from the previously reported monomeric nuclear magnetic resonance (NMR) structure of the CD from the mouse modifier 1 protein (MoMOD1, also known as HP1beta or M31). A prominent pit with a non-polar base is generated at the dimer interface, and is commensurate with binding an extended pentapeptide motif. Sequence alignments based on this structure highlight differences between CDs and CSDs that are superimposed on a common structural core. The analyses also revealed a previously unrecognised circumferential hydrophobic sash around the surface of the CD structure. CONCLUSIONS Dimerisation through the CSD of HP1-like proteins results in the simultaneous formation of a putative protein-protein interaction pit, providing a potential means of targeting CSD-containing proteins to particular chromatin sites.

Different modes of interaction by TIAR and HuR with target RNA and DNA

TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U–rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2′-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways.

Dimerization of Plant Defensin NaD1 Enhances Its Antifungal Activity

Background: NaD1 is a potent antifungal plant defensin from Nicotiana alata flowers. Results: NaD1 forms dimers as determined by x-ray crystallographic, biophysical, and biochemical approaches. Conclusion: Dimerization of NaD1 enhances its fungal cell killing. Significance: Understanding the molecular basis of NaD1 antifungal activity helps define defensin function and has potential application for improving plant resistance against agronomically important fungal pathogens. The plant defensin, NaD1, from the flowers of Nicotiana alata, is a member of a family of cationic peptides that displays growth inhibitory activity against several filamentous fungi, including Fusarium oxysporum. The antifungal activity of NaD1 has been attributed to its ability to permeabilize membranes; however, the molecular basis of this function remains poorly defined. In this study, we have solved the structure of NaD1 from two crystal forms to high resolution (1.4 and 1.58 Å, respectively), both of which contain NaD1 in a dimeric configuration. Using protein cross-linking experiments as well as small angle x-ray scattering analysis and analytical ultracentrifugation, we show that NaD1 forms dimers in solution. The structural studies identified Lys4 as critical in formation of the NaD1 dimer. This was confirmed by site-directed mutagenesis of Lys4 that resulted in substantially reduced dimer formation. Significantly, the reduced ability of the Lys4 mutant to dimerize correlated with diminished antifungal activity. These data demonstrate the importance of dimerization in NaD1 function and have implications for the use of defensins in agribiotechnology applications such as enhancing plant crop protection against fungal pathogens.

Structural Basis for Recognition of High Mannose Type Glycoproteins by Mammalian Transport Lectin VIP36

VIP36 functions as a transport lectin for trafficking certain high mannose type glycoproteins in the secretory pathway. Here we report the crystal structure of VIP36 exoplasmic/luminal domain comprising a carbohydrate recognition domain and a stalk domain. The structures of VIP36 in complex with Ca2+ and mannosyl ligands are also described. The carbohydrate recognition domain is composed of a 17-stranded antiparallel β-sandwich and binds one Ca2+ adjoining the carbohydrate-binding site. The structure reveals that a coordinated Ca2+ ion orients the side chains of Asp131, Asn166, and His190 for carbohydrate binding. This result explains the Ca2+-dependent carbohydrate binding of this protein. The Man-α-1,2-Man-α-1,2-Man, which corresponds to the D1 arm of high mannose type glycan, is recognized by eight residues through extensive hydrogen bonds. The complex structures reveal the structural basis for high mannose type glycoprotein recognition by VIP36 in a Ca2+-dependent and D1 arm-specific manner.

United we stand : combining structural methods

High-resolution techniques are the mainstay of structural biologists; however, to address challenging biological systems many are now turning to hybrid approaches that use complementary structural data. In this review we outline the types of structural problems that benefit from combining results of many methods, we summarise the types of data that can be generated by complementary approaches, and we highlight the application of combined methods in structural biology with recent structural studies of membrane proteins, mega-complexes and inherently flexible proteins.

Cortactin Adopts a Globular Conformation and Bundles Actin into Sheets

Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: An antibiotic target

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug‐resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo‐ and apo‐forms of SaBPL using X‐ray crystallography. We also present small‐angle X‐ray scattering data of SaBPL in complex with its biotin‐carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl‐5′‐AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery.

Evaluating protein:protein complex formation using synchrotron radiation circular dichroism spectroscopy.

Circular dichroism (CD) spectroscopy beamlines at synchrotrons produce dramatically higher light flux than conventional CD instruments. This property of synchrotron radiation circular dichroism (SRCD) results in improved signal‐to‐noise ratios and allows data collection to lower wavelengths, characteristics that have led to the development of novel SRCD applications. Here we describe the use of SRCD to study protein complex formation, specifically evaluating the complex formed between carboxypeptidase A and its protein inhibitor latexin. Crystal structure analyses of this complex and the individual proteins reveal only minor changes in secondary structure of either protein upon complex formation (i.e., it involves only rigid body interactions). Conventional CD spectroscopy reports on changes in secondary structure and would therefore not be expected to be sensitive to such interactions. However, in this study we have shown that SRCD can identify differences in the vacuum ultraviolet CD spectra that are significant and attributable to complex formation. Proteins 2008.

Improved radiation dose efficiency in solution SAXS using a sheath flow sample environment

Coflow is a new method for delivering radiation-sensitive biological and other solution-based samples to high-brightness X-ray beamlines that exploits laminar flow to ameliorate radiation-damage limitations and provides a host of practical improvements associated with these types of experiments.

Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis.

Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, alpha-DsbA1 and alpha-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein alpha-DsbA1; the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of alpha-DsbA1 for both dithiol oxidants (E. coli DsbA, 12%) and disulfide isomerases (E. coli DsbC, 14%) is similar. We therefore sought to establish whether alpha-DsbA1 is an oxidant or an isomerase based on functional activity. The purified alpha-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that alpha-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified alpha-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins.