Helen R Mott

University of Cambridge, Cambridge, England, United Kingdom

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Publications (50)295.89 Total impact

  • Helen R Mott, Darerca Owen
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    ABSTRACT: The Ras superfamily small G proteins are master regulators of a diverse range of cellular processes and act via downstream effector molecules. The first structure of a small G protein-effector complex, that of Rap1A with c-Raf1, was published 20 years ago. Since then, the structures of more than 60 small G proteins in complex with their effectors have been published. These effectors utilize a diverse array of structural motifs to interact with the G protein fold, which we have divided into four structural classes: intermolecular β-sheets, helical pairs, other interactions, and pleckstrin homology (PH) domains. These classes and their representative structures are discussed and a contact analysis of the interactions is presented, which highlights the common effector-binding regions between and within the small G protein families.
    Critical Reviews in Biochemistry and Molecular Biology 04/2015; DOI:10.3109/10409238.2014.999191 · 5.81 Impact Factor
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    ABSTRACT: RalA and RalB are members of the Ras family of small G proteins and are activated downstream of Ras via RalGEFs. The RalGEF-Ral axis represents one of the major effector pathways controlled by Ras and as such is an important pharmacological target. RalA and RalB are approximately 80% identical at the amino acid level; despite this they have distinct roles both in normal cells and in the disease state. We have used our structure of RalB-RLIP76 to guide an analysis of Ral-effector interaction interfaces, creating panels of mutant proteins to probe the energetics of these interactions. The data provide a physical mechanism that underpins the effector selective mutations commonly employed to dissect Ral G protein function. Comparing the energetic landscape of the RalB-RLIP76 and RalB-Sec5 complexes reveals mutations in RalB that differentially bind the two effector proteins. A panel of RLIP76 mutants was used to probe the interaction between RLIP76 and RalA/B. Despite 100% sequence identity in the RalA/B contact residues with RLIP76, differences still exist in the energetic profiles of the two complexes. Therefore we have revealed properties that may account for some of the functional separation observed with RalA and RalB at a cellular level. Our mutations, in both the Ral isoforms and RLIP76, provide new tools that can be employed to parse the complex biology of Ral G protein signalling networks. The combination of this thermodynamic and structural data can also guide efforts to ablate RalA/B activity with small molecules and peptides.
    Biochemistry 01/2015; 54(6). DOI:10.1021/bi501530u · 3.19 Impact Factor
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    ABSTRACT: Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.
    Journal of Biomolecular NMR 01/2015; 61(3-4). DOI:10.1007/s10858-015-9899-6 · 3.31 Impact Factor
  • Helen R Mott, Darerca Owen
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    ABSTRACT: RLIP76 (Ral-interacting protein of 76 kDa) [also known as RalBP1 (Ral-binding protein 1)] is an effector for the Ral family small GTPases. RLIP76 has been implicated in a number of cell processes, including receptor-mediated endocytosis, cell migration, mitochondrial division and metabolite transport. RLIP76 has two recognizable domains in the centre of the protein sequence: a GAP (GTPase-activating protein) domain for the Rho family G-proteins and an RBD (Ral-binding domain). The remainder of RLIP76 has no discernable homology with other proteins. The RBD forms a simple coiled-coil of two α-helices, which interacts with RalB by binding to both of the nucleotide-sensitive 'switch' regions. Both of these RLIP76 helices are involved in the interaction with Ral, but the interhelix loop is left free. This is the location of one of the two ATP-binding sites that have been identified in RLIP76 and suggests that Ral interaction would not prevent ATP binding. The structure of the RhoGAP-RBD dyad shows that the two domains are fixed in their orientation by a relatively rigid linker. This domain arrangement allows the two domains to engage Rho family and Ral small G-proteins simultaneously at the membrane. This suggests that RLIP76 is a node for Rho and Ras family signalling.
    Biochemical Society Transactions 02/2014; 42(1):52-8. DOI:10.1042/BST20130231 · 3.24 Impact Factor
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    ABSTRACT: Protein kinase C-related kinases (PRKs) are members of the protein kinase C superfamily of serine-threonine kinases and can be activated by binding to members of the Rho family of GTPases via a Rho-binding motif known as an HR1 domain. Three tandem HR1 domains reside at the N-terminus of the PRKs. We have assessed the ability of the HR1a and HR1b domains from the three PRK isoforms (PRK1, PRK2, and PRK3) to interact with the three Rho isoforms (RhoA, RhoB, and RhoC). The affinities of RhoA and RhoC for a construct encompassing both PRK1 HR1 domains were similar to those for the HR1a domain alone, suggesting that these interactions are mediated solely by the HR1a domain. The affinities of RhoB for both the PRK1 HR1a domain and the HR1ab didomain were higher than those of RhoA or RhoC. RhoB also bound more tightly to the didomain than to the HR1a domain alone, implicating the HR1b domain in the interaction. As compared with PRK1 HR1 domains, PRK2 and PRK3 domains bind less well to all Rho isoforms. Uniquely, however, the PRK3 domains display a specificity for RhoB that requires both the C-terminus of RhoB and the PRK3 HR1b domain. The thermal stability of the HR1a and HR1b domains was also investigated. The PRK2 HR1a domain was found to be the most thermally stable, while PRK2 HR1b, PRK3 HR1a, and PRK3 HR1b domains all exhibited lower melting temperatures, similar to that of the PRK1 HR1a domain. The lower thermal stability of the PRK2 and PRK3 HR1b domains may impart greater flexibility, driving their ability to interact with Rho isoforms.
    Biochemistry 10/2013; 52(45):7999–8011. DOI:10.1021/bi401216w · 3.19 Impact Factor
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    ABSTRACT: RLIP76 is an effector for Ral small GTPases, which in turn lie downstream of the master regulator Ras. Evidence is growing that Ral and RLIP76 play a role in tumorigenesis, invasion, and metastasis. RLIP76 contains both a RhoGAP domain and a Ral binding domain (GBD) and is, therefore, a node between Ras and Rho family signaling. The structure of the RhoGAP-GBD dyad reveals that the RLIP76 RhoGAP domain adopts a canonical RhoGAP domain structure and that the linker between the two RLIP76 domains is structured, fixing the orientation of the two domains and allowing RLIP76 to interact with Rho-family GTPases and Ral simultaneously. However, the juxtaposed domains do not influence each other functionally, suggesting that the RLIP76-Ral interaction controls cellular localization and that the fixed orientation of the two domains orientates the RhoGAP domain with respect to the membrane, allowing it to be perfectly poised to engage its target G proteins.
    Structure 10/2013; 21(12). DOI:10.1016/j.str.2013.09.007 · 6.79 Impact Factor
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    ABSTRACT: The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale-including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy-we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.
    Proceedings of the National Academy of Sciences 03/2013; DOI:10.1073/pnas.1219476110 · 9.81 Impact Factor
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    ABSTRACT: RLIP76 (also known as RalBP1) is an effector for Ral small G proteins. RLIP76 is a multifunctional, multi-domain protein that includes a GTPase activating domain for the Rho family (RhoGAP domain) and a GTPase binding domain (GBD) for the Ral small G proteins. The juxtaposition of these two domains (GAP and GBD) may be a strategy employed to co-ordinate regulation of Rho family and Ral-controlled signalling pathways at a crossover node. Here we present the 1H, 15N and 13C NMR backbone and sidechain resonance assignments of the GAP and GBD di-domain (31kDa). KeywordsRLIP76–RalBP1–Ral GTPase–Multidomain protein–Endocytosis–Signalling
    Biomolecular NMR Assignments 10/2011; 6(2):1-4. DOI:10.1007/s12104-011-9337-y · 0.82 Impact Factor
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    ABSTRACT: Background / Purpose: Sensory rhodopsin II (pSRII) is an archaeal phototaxis receptor and a member of the seven transmembrane (7TM) class of α-helical membrane proteins. Some common features of pSRII with the important class of G-protein coupled receptors (GPCR) combined with the ability to express labeled proteins in E. coli make it an excellent model system for a possible extension of solution NMR techniques to 7TM proteins. Main conclusion: Complete backbone assignment and the full 3D structure determination by solution NMR has been achieved for detergent-solubilised pSRII, demonstrating the feasibility of structural studies of proteins of this size. The study was expanded with analysis of the backbone dynamics, measurements in more native-like phospholipid bicelles and mapping of protein-detergent interactions.Despite problems of quantity and stability, there are encouraging indications to suggest that, in the near future, heterologous expression systems will provide labeled GPCRs in sufficient quantities to produce NMR samples. Similar NMR methods that we developed for pSRII should be applicable to small- to medium-sized members of the important GPCR family of 7TM proteins.
    Biochemical Society 2011 - Annual Symposium - Recent Advances in Membrane Biochemistry; 02/2011
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    ABSTRACT: Protein kinase C-related kinases (PRKs) are serine/threonine kinases that are members of the protein kinase C superfamily and can be activated by binding to members of the Rho family of small G proteins via a Rho binding motif known as an HR1 domain. The PRKs contain three tandem HR1 domains at their N-termini. The structure of the HR1a domain from PRK1 in complex with RhoA [Maesaki, R., et al. (1999) Mol. Cell 4, 793-803] identified two potential contact interfaces between the G protein and the HR1a domain. In this work, we have used an alanine scanning mutagenesis approach to identify whether both contact sites are used when the two proteins interact in solution and also whether HR1b, the second HR1 domain from PRK1, plays a role in binding to RhoA. The mutagenesis identified just one contact site as being relevant for binding of RhoA and HR1a in solution, and the HR1b domain was found not to contribute to RhoA binding. The folded state and thermal stability of the HR1a and HR1b domains were also investigated. HR1b was found to be more thermally stable than HR1a, and it is hypothesized that the differences in the biophysical properties of these two domains govern their interaction with small G proteins.
    Biochemistry 02/2011; 50(14):2860-9. DOI:10.1021/bi200039u · 3.19 Impact Factor
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    ABSTRACT: RLIP76 (RalBP1) is a multidomain protein that interacts with multiple small G protein families: Ral via a specific binding domain, and Rho and R-Ras via a GTPase activating domain. RLIP76 interacts with endocytosis proteins and has also been shown to behave as a membrane ATPase that transports chemotherapeutic agents from the cell. We have determined the structure of the Ral-binding domain of RLIP76 and show that it comprises a coiled-coil motif. The structure of the RLIP76-RalB complex reveals a novel mode of binding compared to the structures of RalA complexed with the exocyst components Sec5 and Exo84. RLIP76 interacts with both nucleotide-sensitive regions of RalB, and key residues in the interface have been identified using affinity measurements of RalB mutants. Sec5, Exo84, and RLIP76 bind Ral proteins competitively and with similar affinities in vitro.
    Structure 08/2010; 18(8):985-95. DOI:10.1016/j.str.2010.05.013 · 6.79 Impact Factor
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    ABSTRACT: Seven-helix membrane proteins represent a challenge for structural biology. Here we report the first NMR structure determination of a detergent-solubilized seven-helix transmembrane (7TM) protein, the phototaxis receptor sensory rhodopsin II (pSRII) from Natronomonas pharaonis, as a proof of principle. The overall quality of the structure ensemble is good (backbone r.m.s. deviation of 0.48 A) and agrees well with previously determined X-ray structures. Furthermore, measurements in more native-like small phospholipid bicelles indicate that the protein structure is the same as in detergent micelles, suggesting that environment-specific effects are minimal when using mild detergents. We use our case study as a platform to discuss the feasibility of similar solution NMR studies for other 7TM proteins, including members of the family of G protein-coupled receptors.
    Nature Structural & Molecular Biology 06/2010; 17(6):768-74. DOI:10.1038/nsmb.1807 · 11.63 Impact Factor
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    Helen R Mott, Darerca Owen
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    ABSTRACT: RLIP76 (RalBP1) is a multidomain protein that is a downstream effector of the small GTP ases RalA and RalB. As well as the Ral binding domain it contains a RhoGAP domain active against Cdc42 and Rac1. RLIP76 also binds to proteins involved in endocytosis and to R-Ras. We recently solved the structure of the Ral binding domain of RLIP76 and the structure of the complex that it forms with RalB. The structure shows that, unlike the other Ral effectors characterized so far, RLIP76 forms a coiled-coil that interacts with RalB. The RLIP76 Ral binding domain binds to both the switch regions of RalB, which are the parts of the G protein that chance conformation upon nucleotide exchange. Here, we review our structure and discuss how it sheds light on the other functions of RLIP76.
    Small GTPases 01/2010; 1(3):157-160. DOI:10.4161/sgtp.1.3.14233
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    ABSTRACT: Several species of African trypanosomes cause fatal disease in livestock, but most cannot infect humans due to innate trypanosome lytic factors (TLFs). Human TLFs are pore forming high-density lipoprotein (HDL) particles that contain apolipoprotein L-I (apoL-I) the trypanolytic component, and haptoglobin-related protein (Hpr), which binds free hemoglobin (Hb) in blood and facilitates the uptake of TLF via a trypanosome haptoglobin-hemoglobin receptor. The human-infective Trypanosoma brucei rhodesiense escapes lysis by TLF by expression of serum resistance-associated (SRA) protein, which binds and neutralizes apoL-I. Unlike humans, baboons are not susceptible to infection by T. b. rhodesiense due to previously unidentified serum factors. Here, we show that baboons have a TLF complex that contains orthologs of Hpr and apoL-I and that full-length baboon apoL-I confers trypanolytic activity to mice and when expressed together with baboon Hpr and human apoA-I, provides protection against both animal infective and the human-infective T. brucei rhodesiense in vivo. We further define two critical lysines near the C terminus of baboon apoL-1 that are necessary and sufficient to prevent binding to SRA and thereby confer resistance to human-infective trypanosomes. These findings form the basis for the creation of TLF transgenic livestock that would be resistant to animal and human-infective trypanosomes, which would result in the reduction of disease and the zoonotic transmission of human infective trypanosomes.
    Proceedings of the National Academy of Sciences 10/2009; 106(46):19509-14. DOI:10.1073/pnas.0905669106 · 9.81 Impact Factor
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    ABSTRACT: We report (1)H, (13)C and (15)N resonance assignments for Binder of Arl Two (BART), an effector of the small G protein Arl2. The BMRB accession code is 15914.
    Biomolecular NMR Assignments 07/2009; 3(1):33-6. DOI:10.1007/s12104-008-9135-3 · 0.82 Impact Factor
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    ABSTRACT: The small G proteins RalA/B have a crucial function in the regulatory network that couples extracellular signals with appropriate cellular responses. RalA/B are an important component of the Ras signaling pathway and, in addition to their role in membrane trafficking, are implicated in the initiation and maintenance of tumorigenic transformation of human cells. RalA and RalB share 85% sequence identity and collaborate in supporting cancer cell proliferation but have markedly different effects. RalA is important in mediating proliferation, while depletion of RalB results in transformed cells undergoing apoptosis. Crystal structures of RalA in the free form and in complex with its effectors, Sec5 and Exo84, have been solved. Here we have determined the solution structure of free RalB bound to the GTP analogue GMPPNP to an RMSD of 0.6 A. We show that, while the overall architecture of RalB is very similar to the crystal structure of RalA, differences exist in the switch regions, which are sensitive to the bound nucleotide. Backbone 15N dynamics suggest that there are four regions of disorder in RalB: the P-loop, switch I, switch II, and the loop comprising residues 116-121, which has a single residue insertion compared to RalA. 31P NMR data and the structure of RalB.GMPPNP show that the switch regions predominantly adopt state 1 (Ras nomenclature) in the unbound form, which in Ras is not competent to bind effectors. In contrast, 31P NMR analysis of RalB.GTP reveals that conformations corresponding to states 1 and 2 are both sampled in solution and that addition of an effector protein only partially stabilizes state 2.
    Biochemistry 02/2009; 48(10):2192-206. DOI:10.1021/bi802129d · 3.19 Impact Factor
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    ABSTRACT: We report here the (1)H, (15)N and (13)C resonance assignments for the small G protein RalB bound to the GTP analogue, GMPPNP and complexed with the Ral binding domain of its downstream effector RLIP76. The BMRB accession code is 15525.
    Biomolecular NMR Assignments 01/2009; 2(2):179-82. DOI:10.1007/s12104-008-9115-7 · 0.82 Impact Factor
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    ABSTRACT: We report (1)H and (15)N resonance assignments for the free Ral binding domain of RLIP76 (393-446) and the (1)H, (15)N and (13)C resonance assignments for the RLIP76 Ral binding domain in complex with the active conformation of RalB. The BMRB accession code for free RLIP76 is 15524 and in complex with RalB is 15525.
    Biomolecular NMR Assignments 01/2009; 2(2):191-4. DOI:10.1007/s12104-008-9118-4 · 0.82 Impact Factor
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    ABSTRACT: The ADP-ribosylation factor-like (Arl) family of small G proteins are involved in the regulation of diverse cellular processes. Arl2 does not appear to be membrane localized and has been implicated as a regulator of microtubule dynamics. The downstream effector for Arl2, Binder of Arl 2 (BART) has no known function but, together with Arl2, can enter mitochondria and bind the adenine nucleotide transporter. We have solved the solution structure of BART and show that it forms a novel fold composed of six alpha-helices that form three interlocking "L" shapes. Analysis of the backbone dynamics reveals that the protein is highly anisotropic and that the loops between the central helices are dynamic. The regions involved in the binding of Arl2 were mapped onto the surface of BART and are found to localize to these loop regions. BART has faces of differing charge and structural elements, which may explain how it can interact with other proteins.
    Journal of Biological Chemistry 12/2008; 284(2):992-9. DOI:10.1074/jbc.M806167200 · 4.60 Impact Factor

Publication Stats

2k Citations
295.89 Total Impact Points

Institutions

  • 1999–2015
    • University of Cambridge
      • Department of Biochemistry
      Cambridge, England, United Kingdom
  • 1992–1995
    • University of Oxford
      • Department of Biochemistry
      Oxford, England, United Kingdom