62 results found
Jamshidiha M, Lanyon-Hogg T, Sutherell C, et al., 2021, Identification of the first structurally validated covalent ligands of the small GTPase RAB27A, RSC Medicinal Chemistry, Vol: 13, Pages: 150-155, ISSN: 2632-8682
Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane via protein–protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket (‘WF-binding pocket’) via a conserved tryptophan–phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets.
Saunders CN, Cota E, Baum J, et al., 2020, Peptide probes for Plasmodium falciparum MyoA tail interacting protein (MTIP): exploring the druggability of the malaria parasite motor complex, ACS Chemical Biology, Vol: 15, Pages: 1313-1320, ISSN: 1554-8929
Malaria remains an endemic tropical disease, and the emergence of Plasmodium falciparum parasites resistant to current front-line medicines means that new therapeutic targets are required. The Plasmodium glideosome is a multiprotein complex thought to be essential for efficient host red blood cell invasion. At its core is a myosin motor, Myosin A (MyoA), which provides most of the force required for parasite invasion. Here, we report the design and development of improved peptide-based probes for the anchor point of MyoA, the P. falciparum MyoA tail interacting protein (PfMTIP). These probes combine low nanomolar binding affinity with significantly enhanced cell penetration and demonstrable competitive target engagement with native PfMTIP through a combination of Western blot and chemical proteomics. These results provide new insights into the potential druggability of the MTIP/MyoA interaction and a basis for the future design of inhibitors.
Spanu P, Przydacz M, Jones R, et al., 2020, Mode of action of the catalytic site in the N-terminal ribosome-inactivating domain of barley JIP60, Plant Physiology, ISSN: 0032-0889
Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein from barley (Hordeum vulgare) and is involved in the plant immune response dependent on the jasmonate hormones. Here, we demonstrate that transient expression in Nicotiana benthamiana of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163 to 185) is removed, initiates cell death leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in N. benthamiana, as well as through an in vitro translation-inactivation assay. We found that Tyrosine 96, Glutamate 202, Arginine 205, and Tryptophan 235 in the presumptive active site of JIP60 are conserved in 815 plant ribosome inactivating proteins (RIP) in the Pfam database identified using HMMER as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose a role for these amino acids in JIP60 activity (depurination of adenosine in ribosomes). This study provides insight into the catalytic mechanism of this protein.
Dian C, Inmaculada P-D, Riviere F, et al., 2020, High-resolution snapshots of human N-myristoyltransferase in action illuminate a mechanism promoting N-terminal Lys and Gly myristoylation, Nature Communications, Vol: 11, Pages: 1-15, ISSN: 2041-1723
The promising drug target N-myristoyltransferase (NMT) catalyses an essential protein modification thought to occur exclusively at N-terminal glycines (Gly). Here, we present high-resolution human NMT1 structures co-crystallised with reactive cognate lipid and peptide substrates, revealing high-resolution snapshots of the entire catalytic mechanism from the initial to final reaction states. Structural comparisons, together with biochemical analysis, provide unforeseen details about how NMT1 reaches a catalytically competent conformation in which the reactive groups are brought into close proximity to enable catalysis. We demonstrate that this mechanism further supports efficient and unprecedented myristoylation of an N-terminal lysine side chain, providing evidence that NMT acts both as N-terminal-lysine and glycine myristoyltransferase.
Przydacz M, Bruderer M, Belmans G, et al., 2019, Structure/function determination of JIP60 mode of action: The ribosome inactivating protein targeted by RNase-like powdery mildew effectors, 18th Congress of International-Society-for-Molecular-Plant-Microbe-Interactions (IS-MPMI), Publisher: AMER PHYTOPATHOLOGICAL SOC, Pages: 165-165, ISSN: 0894-0282
Pennington HG, Rhian J, Kwon S, et al., 2019, The fungal ribonuclease-like effector protein CSEP0064/BEC1054 represses plant immunity and interferes with degradation of host ribosomal RNA, PLoS Pathogens, Vol: 15, ISSN: 1553-7366
The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus.
Jamshidiha M, Pérez-Dorado I, Murray JW, et al., 2019, Coping with strong translational noncrystallographic symmetry and extreme anisotropy in molecular replacement with Phaser: human Rab27a, Acta Crystallographica Section D Structural Biology, Vol: 75, Pages: 342-353, ISSN: 2059-7983
Data pathologies caused by effects such as diffraction anisotropy and translational noncrystallographic symmetry (tNCS) can dramatically complicate the solution of the crystal structures of macromolecules. Such problems were encountered in determining the structure of a mutant form of Rab27a, a member of the Rab GTPases. Mutant Rab27a constructs that crystallize in the free form were designed for use in the discovery of drugs to reduce primary tumour invasiveness and metastasis. One construct, hRab27a<jats:sup>Mut</jats:sup>, crystallized within 24 h and diffracted to 2.82 Å resolution, with a unit cell possessing room for a large number of protein copies. Initial efforts to solve the structure using molecular replacement by <jats:italic>Phaser</jats:italic> were not successful. Analysis of the data set revealed that the crystals suffered from both extreme anisotropy and strong tNCS. As a result, large numbers of reflections had estimated standard deviations that were much larger than their measured intensities and their expected intensities, revealing problems with the use of such data at the time in <jats:italic>Phaser</jats:italic>. By eliminating extremely weak reflections with the largest combined effects of anisotropy and tNCS, these problems could be avoided, allowing a molecular-replacement solution to be found. The lessons that were learned in solving this structure have guided improvements in the numerical analysis used in <jats:italic>Phaser</jats:italic>, particularly in identifying diffraction measurements that convey very little information content. The calculation of information content could also be applied as an alternative to ellipsoidal truncation. The post-mortem analysis also revealed an oversight in accounting for measurement errors in the fast rotation function. While the crystal of mutant Rab27a is not amenable to drug screening, the structure can guide new modifications to obtain more sui
Rasheed M, Jamshidiha M, Puglisi R, et al., 2019, Structural and functional characterization of a frataxin from a thermophilic organism, FEBS Journal, Vol: 286, Pages: 495-506, ISSN: 0014-2956
Frataxins form an interesting family of iron‐binding proteins with an almost unique fold and are highly conserved from bacteria to primates. They have a pivotal role in iron–sulfur cluster biogenesis as regulators of the rates of cluster formation, as it is testified by the fact that frataxin absence is incompatible with life and reduced levels of the protein lead to the recessive neurodegenerative disease Friedreich's ataxia. Despite its importance, the structure of frataxin has been solved only from relatively few species. Here, we discuss the X‐ray structure of frataxin from the thermophilic fungus Chaetomium thermophilum, and the characterization of its interactions and dynamics in solution. We show that this eukaryotic frataxin has an unusual variation in the classical frataxin fold: the last helix is shorter than in other frataxins which results in a less symmetrical and compact structure. The stability of this protein is comparable to that of human frataxin, currently the most stable among the frataxin orthologues. We also characterized the iron‐binding mode of Ct frataxin and demonstrated that it binds it through a semiconserved negatively charged ridge on the first helix and beta‐strand. Moreover, this frataxin is also able to bind the bacterial ortholog of the desulfurase, which is central in iron–sulfur cluster synthesis, and act as its inhibitor.
Yu J, Knoppova J, Michoux F, et al., 2018, Ycf48 involved in the biogenesis of the oxygen-evolving photosystem II complex is a seven-bladed beta-propeller protein, Proceedings of the National Academy of Sciences, Vol: 115, Pages: E7824-E7833, ISSN: 0027-8424
Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved “Arg patch” on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.
Riviere F, Dian C, Perez-Dorado I, et al., 2018, Mechanistic insight into HsNMT1-mediated acylation, Publisher: WILEY, Pages: 421-422, ISSN: 2211-5463
Zhao W, Jamshidiha M, Lanyon-Hogg T, et al., 2017, Direct targeting of the ras GTPase superfamily through structure-based design, Current Topics in Medicinal Chemistry, Vol: 17, Pages: 16-29, ISSN: 1568-0266
The Ras superfamily of small monomeric GTPases includes some of the most prominent cancer targets for which no selective therapeutic agent has yet been successfully developed. The turn of the millennium saw a resurgence of efforts to target these enzymes using new and improved biophysical techniques to overcome the perceived difficulties of insurmountably high affinity for guanosine nucleotides and flat, flexible topology lacking suitable pockets for small molecule inhibitors. Further, recent investigations have begun to probe the dynamic conformational status of GTP-bound Ras, opening up new mechanisms of inhibition. While much of the literature has focused on the oncogenic Ras proteins, particularly K-Ras, these represent only a small minority of therapeutically interesting targets within the superfamily; for example, the Rab GTPases are the largest subfamily of about 70 members, and present an as yet untapped class of potential targets. The present review documents the key methodologies employed to date in structure-guided attempts to drug the Ras GTPases, and forecasts their transferability to other similarly challenging proteins in the superfamily.
Perez-Dorado JI, Chee SM, Hale L, et al., 2016, Unravelling the molecular basis of adhesion to the host via Als1, a major virulence factor from Candida albicans, 41st FEBS Congress on Molecular and Systems Biology for a Better Life, Publisher: WILEY-BLACKWELL, Pages: 78-78, ISSN: 1742-464X
Hagiwara Y, Sieverling L, Hanif F, et al., 2016, Consequences of point mutations in melanoma-associated antigen 4 (MAGE-A4) protein: Insights from structural and biophysical studies., Scientific Reports, Vol: 6, ISSN: 2045-2322
The Melanoma-Associated Antigen A4 (MAGE-A4) protein is a target for cancer therapy. The function of this protein is not well understood. We report the first comprehensive study on key cancer-associated MAGE-A4 mutations and provide analysis on the consequences of these mutations on the structure, folding and stability of the protein. Based on Nuclear Magnetic Resonance and Circular Dichroism, these mutations had no significant effects on the structure and the folding of the protein. Some mutations affected the thermal stability of the protein remarkably. Native mass spectrometry of wild-type MAGE-A4 showed a broad charge state distribution suggestive of a structurally dynamic protein. Significant intensity was found in relatively low charge states, indicative of a predominantly globular form and some population in more extended states. The latter is supported by Ion Mobility measurements. The MAGE-A4 mutants exhibited similar features. These novel molecular insights shed further light on better understanding of these proteins, which are implicated in a wide range of human cancers.
Moyes DL, Wilson D, Richardson JP, et al., 2016, Candidalysin is a fungal peptide toxin critical for mucosal infection, Nature, Vol: 532, Pages: 64-68, ISSN: 0028-0836
Hoyer LL, Cota E, 2016, Candida albicans agglutinin-like sequence (aIs) family vignettes: a review of aIs protein structure and function, Frontiers in Microbiology, Vol: 7, ISSN: 1664-302X
Approximately two decades have passed since the description of the first gene inthe Candida albicans ALS (agglutinin-like sequence) family. Since that time, much hasbeen learned about the composition of the family and the function of its encoded cellsurfaceglycoproteins. Solution of the structure of the Als adhesive domain providesthe opportunity to evaluate the molecular basis for protein function. This review articleis formatted as a series of fundamental questions and explores the diversity of the Alsproteins, as well as their role in ligand binding, aggregative effects, and attachment toabiotic surfaces. Interaction of Als proteins with each other, their functional equivalence,and the effects of protein abundance on phenotypic conclusions are also examined.Structural features of Als proteins that may facilitate invasive function are considered.Conclusions that are firmly supported by the literature are presented while highlightingareas that require additional investigation to reveal basic features of the Als proteins,their relatedness to each other, and their roles in C. albicans biology.
Cota E, Hoyer LL, 2015, The Candida albicans agglutinin-like sequence family of adhesins: functional insights gained from structural analysis, Future Microbiology, Vol: 10, Pages: 1635-1648, ISSN: 1746-0921
Candida albicans colonizes many host sites suggesting its interaction with diverse ligands.Candida albicans adhesion is mediated by a number of proteins including those in the Als(agglutinin-like sequence) family, which have been studied intensively. The recent solutionof the Als binding domain structure ended years of speculation regarding the molecularmechanism for Als adhesive function. Als adhesins bind flexible C termini from a broadcollection of proteins, providing the basis for adhesion to various cell types and perhaps forC. albicans broad tissue tropism. Understanding adhesive functions at the molecular level willreveal the sequence of events in C. albicans pathogenesis, from host recognition to complexinteractions such as development of polymicrobial biofilms or disseminated disease.
Yusuf NA, Green JL, Wall RJ, et al., 2015, The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain, Journal of Biological Chemistry, Vol: 290, Pages: 12147-12164, ISSN: 1083-351X
Myosin B (MyoB) is one of the two short class XIV myosinsencoded in the Plasmodium genome. Class XIV myosins arecharacterized by a catalytic “head,” a modified “neck,” and theabsence of a “tail” region. Myosin A (MyoA), the other class XIVmyosin in Plasmodium, has been established as a component ofthe glideosome complex important in motility and cell invasion,but MyoB is not well characterized. We analyzed the propertiesof MyoB using three parasite species as follows: Plasmodiumfalciparum, Plasmodium berghei, and Plasmodium knowlesi.MyoB is expressed in all invasive stages (merozoites, ookinetes,and sporozoites) of the life cycle, and the protein is found in adiscrete apical location in these polarized cells. In P. falciparum,MyoB is synthesized very late in schizogony/merogony, and itslocation in merozoites is distinct from, and anterior to, that of arange of known proteins present in the rhoptries, rhoptry neckor micronemes. Unlike MyoA, MyoB is not associated withglideosome complex proteins, including the MyoA light chain,myosin A tail domain-interacting protein (MTIP). A uniqueMyoB light chain (MLC-B) was identified that contains a calmodulin-likedomain at the C terminus and an extended N-terminalregion. MLC-B localizes to the same extreme apical polein the cell as MyoB, and the two proteins form a complex. Wepropose that MLC-B is a MyoB-specific light chain, and for theshort class XIV myosins that lack a tail region, the atypical myosinlight chains may fulfill that role.
Douse CH, Vrielink N, Zhang W, et al., 2015, Targeting a Dynamic Protein-Protein Interaction: Fragment Screening against the Malaria Myosin A Motor Complex, CHEMMEDCHEM, Vol: 10, Pages: 134-143, ISSN: 1860-7179
- Author Web Link
- Open Access Link
- Citations: 12
Hoyer LL, Oh S-H, Jones R, et al., 2014, A proposed mechanism for the interaction between the Candida albicans Als3 adhesin and streptococcal cell wall proteins, Frontiers in Microbiology, Vol: 5, ISSN: 1664-302X
C. albicans binds various bacteria, including the oral commensal Streptococcus gordonii. Published reports documented the role of C. albicans Als3 and S. gordonii SspB in this interaction, and the importance of the Als N-terminal domain (NT-Als) in C. albicans adhesion. Here, we demonstrate that Als1 also binds S. gordonii. We also describe use of the NT-Als crystal structure to design mutations that precisely disrupt peptide-binding cavity (PBC) or amyloid-forming region (AFR) function in Als3. C. albicans displaying Als3 PBC mutant proteins showed significantly reduced binding to S. gordonii; mutation of the AFR did not affect the interaction. These observations present an enigma: the Als PBC binds free C termini of ligands, but the SspB C terminus is covalently linked to peptidoglycan and thus unavailable as a ligand. These observations and the predicted SspB elongated structure suggest that partial proteolysis of streptococcal cell wall proteins is necessary for recognition by Als adhesins.
Douse CH, Maas SJ, Thomas JC, et al., 2014, Crystal Structures of Stapled and Hydrogen Bond Surrogate Peptides Targeting a Fully Buried Protein-Helix Interaction, ACS CHEMICAL BIOLOGY, Vol: 9, Pages: 2204-2209, ISSN: 1554-8929
- Author Web Link
- Open Access Link
- Citations: 37
Berry AA, Yang Y, Pakharukova N, et al., 2014, Structural Insight into Host Recognition by Aggregative Adherence Fimbriae of Enteroaggregative Escherichia coli, PLOS PATHOGENS, Vol: 10, ISSN: 1553-7366
- Author Web Link
- Open Access Link
- Citations: 28
Lin J, Oh S-H, Jones R, et al., 2014, The peptide-binding cavity Is essential for Als3-mediated adhesion of Candida albicans to human cells, Journal of Biological Chemistry, Vol: 289, Pages: 18401-18412, ISSN: 1083-351X
Background: Of the eight cell surface glycoproteins in the C. albicans Als family, Als3 makes the largest contribution to adhesion to human cells.Results: Mutation of the Als3 peptide-binding cavity (PBC) results in loss of Als3 adhesive function.Conclusion: The PBC is required for Als3 adhesive function.Significance: Interfering with PBC function is a viable strategy for inhibiting C. albicans adhesion.
Douse CH, Green JL, Salgado PS, et al., 2012, Regulation of the Plasmodium motor complex: phosphorylation of myosin A tail-interacting protein (MTIP) loosens its grip on MyoA., Journal of Biological Chemistry, Vol: 287, Pages: 36968-36977, ISSN: 1083-351X
Background: Recent phosphoproteome data reveal the extent of post-translational phosphorylation in selected apicomplexanparasites.Results: Binding site mutants that mimic the effect of MTIP phosphorylation in vivo severely decrease MyoA binding.Conclusion: Phosphorylation of selected binding site residues modulates the activity of the actomyosin motor.Significance: Study of Apicomplexa phosphosites can inform on the regulation of functions involved in pathogenesis.
James E, Liu M, Sheppard C, et al., 2012, Structural and Mechanistic Basis for the Inhibition of Escherichia coli RNA Polymerase by T7 Gp2, MOLECULAR CELL, Vol: 47, Pages: 755-766, ISSN: 1097-2765
- Author Web Link
- Citations: 34
Moyes DL, Murciano C, Tang S, et al., 2012, Epithelial responses to Candida albicans, MYCOSES, Vol: 55, Pages: 2-3, ISSN: 0933-7407
Liu B, Garnett JA, Lee W-C, et al., 2012, Promoting crystallisation of the Salmonella enteritidis fimbriae 14 pilin SefD using deuterium oxide, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol: 421, Pages: 208-213, ISSN: 0006-291X
- Author Web Link
- Citations: 2
Simpson PJ, Cota E, Bolanos-Garcia VM, 2012, H-1, C-13 and N-15 resonance assignments of the kinetochore localisation domain of BUBR1, a central component of the spindle assembly checkpoint, BIOMOLECULAR NMR ASSIGNMENTS, Vol: 6, Pages: 115-118, ISSN: 1874-2718
- Author Web Link
- Citations: 1
Garnett JA, Martinez-Santos VI, Saldana Z, et al., 2012, Structural insights into the biogenesis and biofilm formation by the Escherichia coli common pilus, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 109, Pages: 3950-3955, ISSN: 0027-8424
- Author Web Link
- Citations: 45
Garnett JA, Simpson PJ, Taylor J, et al., 2012, Structural insight into the role of Streptococcus parasanguinis Fap1 within oral biofilm formation, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol: 417, Pages: 421-426, ISSN: 0006-291X
- Author Web Link
- Citations: 20
Cota E, 2012, Candida albicans Als adhesins - New insights from recent structural and functional data, 11th ASM Conference on Candida and Candidiasis
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