Biomedical subjects

Carol V Robinson

Publications and source records attributed to Carol V Robinson.

At least 19 recordsLinked to original sources

PubMed2025

Uncovering hidden protein modifications with native top-down mass spectrometry.

Protein modifications drive dynamic cellular processes by modulating biomolecular interactions, yet capturing these modifications within their native structural context remains a significant challenge. Native top-down mass spectrometry promises to preserve the critical link between modifications and interactions. However, current methods often fail to detect uncharacterized or low-abundance modifications, limiting insights into proteoform diversity. To address this gap, we introduce precise and accurate Identification Of Native proteoforms (precisION), an interactive end-to-end software package that leverages a robust, data-driven fragment-level open search to detect, localize and quantify 'hidden' modifications within intact protein complexes. Applying precisION to four therapeutically relevant targets-PDE6, ACE2, osteopontin (SPP1) and a GABA transporter (GAT1)-we discover undocumented phosphorylation, glycosylation and lipidation, and resolve previously uninterpretable density in an electron cryo-microscopy map of GAT1. As an open-source software package, precisION offers an intuitive means for interpreting complex protein fragmentation data. This tool will empower the community to unlock the potential of native top-down mass spectrometry, advancing integrative structural biology, molecular pathology and drug development.

Mass Spectrometry↗

PubMed2007

Structural insights into the p97-Ufd1-Npl4 complex.

p97/VCP (Cdc48 in yeast) is an essential and abundant member of the AAA+ family of ATPases and is involved in a number of diverse cellular pathways through interactions with different adaptor proteins. The two most characterized adaptors for p97 are p47 and the Ufd1 (ubiquitin fusion degradation 1)-Npl4 (nuclear protein localization 4) complex. p47 directs p97 to membrane fusion events and has been shown to be involved in protein degradation. The Ufd1-Npl4 complex directs p97 to an essential role in endoplasmic reticulum-associated degradation and an important role in mitotic spindle disassembly postmitosis. Here we describe the structural features of the Ufd1-Npl4 complex and its interaction with p97 with the aid of EM and other biophysical techniques. The Ufd1-Npl4 heterodimer has an elongated bilobed structure that is approximately 80 x 30 A in dimension. One Ufd1-Npl4 heterodimer is shown to interact with one p97 hexamer to form the p97-Ufd1-Npl4 complex. The Ufd1-Npl4 heterodimer emanates from one region on the periphery of the N-D1 plane of the p97 hexamer. Intriguingly, the p97-p47 and the p97-Ufd1-Npl4 complexes are significantly different in stoichiometry, symmetry, and quaternary arrangement, reflecting their specific actions and their ability to interact with additional cofactors that cooperate with p97 in diverse cellular pathways.

Adenosine Triphosphatases↗

PubMed2007

Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity.

AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases.

Animals↗

PubMed2006

Structural basis for protein-protein interactions in the 14-3-3 protein family.

The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein-protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners.

14-3-3 Proteins↗

PubMed2006

Plant transformation by Agrobacterium tumefaciens: modulation of single-stranded DNA-VirE2 complex assembly by VirE1.

Agrobacterium tumefaciens infects plant cells by the transfer of DNA. A key factor in this process is the bacterial virulence protein VirE2, which associates stoichiometrically with the transported single-stranded (ss) DNA molecule (T-strand). As observed in vitro by transmission electron microscopy, VirE2-ssDNA readily forms an extended helical complex with a structure well suited to the tasks of DNA protection and nuclear import. Here we have elucidated the role of the specific molecular chaperone VirE1 in regulating VireE2-VirE2 and VirE2-ssDNA interactions. VirE2 alone formed functional filamentous aggregates capable of ssDNA binding. In contrast, co-expression with VirE1 yielded monodisperse VirE1-VirE2 complexes. Cooperative binding of VirE2 to ssDNA released VirE1, resulting in a controlled formation mechanism for the helical complex that is further promoted by macromolecular crowding. Based on this in vitro evidence, we suggest that the constrained volume of the VirB channel provides a natural site for the exchange of VirE2 binding from VirE1 to the T-strand.

Agrobacterium tumefaciens↗

PubMed2006

The HC fragment of tetanus toxin forms stable, concentration-dependent dimers via an intermolecular disulphide bond.

Protein oligomerisation is a prerequisite for the toxicity of a number of bacterial toxins. Examples include the pore-forming cytotoxin streptolysin O, which oligomerises to form large pores in the membrane and the protective antigen of anthrax toxin, where a heptameric complex is essential for the delivery of lethal factor and edema factor to the cell cytosol. Binding of the clostridial neurotoxins to receptors on neuronal cells is well characterised, but little is known regarding the quaternary structure of these toxins and the role of oligomerisation in the intoxication process. We have investigated the oligomerisation of the receptor binding domain (H(C)) of tetanus toxin, which retains the binding and trafficking properties of the full-length toxin. Electrophoresis, size exclusion chromatography and mass spectrometry were used to demonstrate that H(C) undergoes concentration-dependent oligomerisation in solution. Reducing agents were found to affect H(C) oligomerisation and, using mutagenesis, Cys869 was shown to be essential for this process. Furthermore, the oligomeric state and quaternary structure of H(C) in solution was assessed using synchrotron small-angle X-ray scattering. Ab initio shape analysis and rigid body modelling coupled with mutagenesis data allowed the construction of an unequivocal model of dimeric H(C) in solution. We propose a possible mechanism for H(C) oligomerisation and discuss how this may relate to toxicity.

Binding Sites↗

PubMed2006

Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes.

It is established that noncovalent complexes can be maintained both during and after electrospray and that assemblies of increasing size and complexity often lead to broadened peaks in mass spectra. This broadening arises from the tendency of large protein assemblies to form adducts with salts and is compounded when complexes are isolated directly from cells, without the full protein complement. To investigate the origins of this broadening in mass spectral peaks and to develop the optimal method for analyzing mass spectra of large protein complexes, we have carried out a systematic investigation of a series of noncovalent complexes representing a range of different sizes and architectures. We establish a positive correlation between peak width and the increased mass observed and show that this correlation is independent of the instrumental parameters employed. Using this relationship we show that we can determine masses of both 30S subunits and intact 2.3 MDa 70S ribosomes from Thermus thermophilus. The masses of both particles are consistent with multiple populations of ribosomes. To identify these various populations we combine simulated mass spectra of ribosomes, with and without the full protein complement, and estimate the extent of adducts from our study of known complexes. The results allow us to determine the contribution of the different subpopulations to the overall mass spectrum. We confirm the existence of these subpopulations using tandem mass spectrometry of intact 30S subunits. Overall, the results show that, rather than uniform particles, gas-phase ribosomes consist of a number of discrete populations. More generally, the results establish a rigorous procedure for accurate mass measurement and spectral analysis of heterogeneous macromolecular assemblies.

Models, Molecular↗

PubMed2006

Structure of an Hsp90-Cdc37-Cdk4 complex.

Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle.

Cell Cycle Proteins↗

PubMed2006

Aspects of native proteins are retained in vacuum.

It is now well established that protein complexes can be maintained in the gas phase, under the reduced pressure conditions experienced within mass spectrometers. This remains an intriguing phenomenon, given the importance placed on water molecules in stabilizing protein structure. As a consequence, the overall structure of protein molecules and their complexes in the gas phase continues to stimulate much research. The eventual outcomes of this research will not only increase the number of applications of mass spectrometry to structural biology, but will also enhance our understanding of the interactions important for maintaining protein complexes in the absence of bulk solvent.

Mass Spectrometry↗

PubMed2006

Activation of ubiquitin ligase SCF(Skp2) by Cks1: insights from hydrogen exchange mass spectrometry.

Skp2 is the substrate recognition subunit of the multi-subunit ubiquitin ligase SCF(Skp2). It consists of an N-terminal F-box domain that binds to the Skp1 subunit and thereby tethers it to the SCF catalytic core, and an elongated C-terminal domain comprising ten Leucine-rich repeats (LRR) that binds the substrate. A small accessory protein, Cks1, is required for SCF(Skp2) to target certain substrates, including the Cyclin-dependent kinase inhibitor p27. Here we have used hydrogen/deuterium exchange monitored by mass spectrometry to investigate the mode of action of Cks1 on SCF(Skp2). We show that complex formation between Cks1 and Skp2 causes conformational changes in both proteins in regions distant from the respective binding sites. We find that Skp2 interacts with a localised region of Cks1 but the interaction causes a global change in the hydrogen exchange behaviour of Cks1. Also, whilst Cks1 binds to the most C-terminal LRRs of the elongated Skp2 molecule, the interaction induces conformational changes at the distant N-terminal LRRs, close to the F-box motif. Further, binding of Cks1 to Skp2 significantly stabilises the interaction between Skp2 and Skp1. The results reveal that the C-terminal substrate recognition region of Skp2 is coupled to the N-terminal Skp1-binding region and thereby to the SCF catalytic core; this result adds to the model proposed previously that, whilst the principal function of the F-box protein is to recruit the substrate, an additional function may be to help position the substrate in an optimal way within the SCF complex to enable efficient ubiquitin transfer.

CDC2-CDC28 Kinases↗

PubMed2006

Structural role of Sfi1p-centrin filaments in budding yeast spindle pole body duplication.

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p-centrin complexes containing several repeats show Sfi1p as an alpha helix with centrins wrapped around each repeat and similar centrin-centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p-centrin complex with 15 Sfi1 repeats and 15 centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous alpha helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p-centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly.

Binding Sites↗

PubMed2006

Subunit architecture of multimeric complexes isolated directly from cells.

Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.

Chromatography, Affinity↗

PubMed2006

The component polypeptide chains of bovine insulin nucleate or inhibit aggregation of the parent protein in a conformation-dependent manner.

Amyloid fibrils are typically rigid, unbranched structures with diameters of approximately 10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease and type II diabetes. Insulin is a small, predominantly alpha-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low pH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to cross-seed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of full-length insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underlying protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression.

Amyloid↗

PubMed2006

Targeting C-reactive protein for the treatment of cardiovascular disease.

Complement-mediated inflammation exacerbates the tissue injury of ischaemic necrosis in heart attacks and strokes, the most common causes of death in developed countries. Large infarct size increases immediate morbidity and mortality and, in survivors of the acute event, larger non-functional scars adversely affect long-term prognosis. There is thus an important unmet medical need for new cardioprotective and neuroprotective treatments. We have previously shown that human C-reactive protein (CRP), the classical acute-phase protein that binds to ligands exposed in damaged tissue and then activates complement, increases myocardial and cerebral infarct size in rats subjected to coronary or cerebral artery ligation, respectively. Rat CRP does not activate rat complement, whereas human CRP activates both rat and human complement. Administration of human CRP to rats is thus an excellent model for the actions of endogenous human CRP. Here we report the design, synthesis and efficacy of 1,6-bis(phosphocholine)-hexane as a specific small-molecule inhibitor of CRP. Five molecules of this palindromic compound are bound by two pentameric CRP molecules, crosslinking and occluding the ligand-binding B-face of CRP and blocking its functions. Administration of 1,6-bis(phosphocholine)-hexane to rats undergoing acute myocardial infarction abrogated the increase in infarct size and cardiac dysfunction produced by injection of human CRP. Therapeutic inhibition of CRP is thus a promising new approach to cardioprotection in acute myocardial infarction, and may also provide neuroprotection in stroke. Potential wider applications include other inflammatory, infective and tissue-damaging conditions characterized by increased CRP production, in which binding of CRP to exposed ligands in damaged cells may lead to complement-mediated exacerbation of tissue injury.

Animals↗

PubMed2006

Functional and structural characterization of the Methanosarcina mazei proteasome and PAN complexes.

We have cloned the proteasome and the proteasome activating nucleotidase (PAN) genes from the mesophilic archaeon Methanosarcina mazei and produced the respective proteins in Escherichia coli cultures. The recombinant complexes were purified to homogeneity and characterized biochemically, structurally, and by mass spectrometry. We found that the degradation of Bodipy-casein by Methanosarcina proteasomes was activated by Methanosarcina PAN. Notably, the Methanosarcina PAN unfolded GFP-SsrA only in the presence of Methanosarcina proteasomes. Structural analysis by 2D averaging electron microscopy of negatively stained complexes displayed the typical structure for the proteasome, namely four-striped side-views and sevenfold-symmetric top-views, with 15 nm height and 11 nm diameter. The structural analysis of the PAN preparation revealed also four-striped side-views, albeit with a height of 18 nm and sixfold-symmetric top-views with a diameter of 15 nm, which corresponds most likely to a dimer of two hexameric complexes. Mass spectrometric analysis of both the Methanosarcina and the Methanocaldococcus PAN proteins indicated hexameric complexes. In summary, we performed a functional and structural characterization of the PAN and proteasome complexes from the archaeon M. mazei and described unique new structural and functional features.

Adenosine Triphosphatases↗

PubMed2006

Tandem mass spectrometry of intact GroEL-substrate complexes reveals substrate-specific conformational changes in the trans ring.

It has been suggested that the bacterial GroEL chaperonin accommodates only one substrate at any given time, due to conformational changes to both the cis and trans ring that are induced upon substrate binding. Using electrospray ionization mass spectrometry, we show that indeed GroEL binds only one molecule of the model substrate Rubisco. In contrast, the capsid protein of bacteriophage T4, a natural GroEL substrate, can occupy both rings simultaneously. As these substrates are of similar size, the data indicate that each substrate induces distinct conformational changes in the GroEL chaperonin. The distinctive binding behavior of Rubisco and the capsid protein was further investigated using tandem mass spectrometry on the intact 800-914 kDa GroEL-substrate complexes. Our data suggest that even in the gas phase the substrates remain bound inside the GroEL cavity. The analysis revealed further that binding of Rubisco to the GroEL oligomer stabilizes the chaperonin complex significantly, whereas binding of one capsid protein did not have the same effect. However, addition of a second capsid protein molecule to GroEL resulted in a similar stabilizing effect to that obtained after the binding of a single Rubisco. On the basis of the stoichiometry of the GroEL chaperonin-substrate complex and the dissociation behavior of the two different substrates, we hypothesize that the binding of a single capsid polypeptide does not induce significant conformational changes in the GroEL trans ring, and hence the unoccupied GroEL ring remains accessible for a second capsid molecule.

Bacteriophage T4↗

PubMed2006

Mass spectrometry of macromolecular assemblies: preservation and dissociation.

Mass spectrometry not only plays a crucial role in the identification of proteins involved in the intricate interaction networks of the cell, but also is increasingly involved in the characterization of the non-covalent complexes formed by interacting partners. Recent developments have enabled the use of gas phase dissociation to probe oligomeric organization and topology, and increased understanding of the electrospray process is leading to knowledge of the structure of protein assemblies both in solution and in the gas phase.

Multiprotein Complexes↗

PubMed2006

Structural basis for the inhibition of activin signalling by follistatin.

The secreted, multidomain protein follistatin binds activins with high affinity, inhibiting their receptor interaction. We have dissected follistatin's domain structure and shown that the minimal activin-inhibiting fragment of follistatin is comprised of the first and second Fs domains (Fs12). This protein can bind to activin dimer and form a stable complex containing two Fs12 molecules and one activin dimer. We have solved crystal structures of activin A alone and its complex with Fs12 fragment to 2 A resolution. The complex structure shows how Fs12 molecules wrap around the back of the 'wings' of activin, blocking the type II receptor-binding site on activin A. Arginine 192 in Fs2 is a key residue in this interaction, inserting itself in between activin's fingers. Complex formation imposes a novel orientation for the EGF- and Kazal-like subdomains in the Fs2 domain and activin A shows further variation from the canonical TGF-beta family fold. The structure provides a detailed description of the inhibitory mechanism and gives insights into interactions of follistatin with other TGF-beta family proteins.

Activin Receptors, Type II↗