Biology subjects

Ferguson, J. A.

Publications and source records attributed to Ferguson, J. A..

6 recordsLinked to original sources

bioRxiv2024

Structural and immunological characterization of the H3 influenza hemagglutinin during antigenic drift

The quest for a universal influenza vaccine holds great promise for mitigating the global burden of influenza-related morbidity and mortality. However, challenges persist in identifying conserved epitopes capable of inducing protection. In this study, we explore the influence of glycan evolution on H3 hemagglutinin from 1968 to present day and its impacts on antigenicity and immunogenicity. We observe that the appearance of potential N-linked glycosylation sites in Sing/16 hemagglutinin head domain reduces the binding of broadly neutralizing antibodies and shifts the polyclonal immune response upon vaccination to target the stem. Furthermore, structural characterization of HK/68 and Sing/16 by cryo-electron microscopy shows that while HK/68 is resistant to enzymatic deglycosylation, removal of glycans destabilizes the hyperglycosylated head and membrane-proximal region in Sing/16. These insights expand our understanding of glycans beyond their role in protein folding and highlight the interplay among glycan integration and immune recognition to design a universal influenza vaccine.

immunology↗

bioRxiv2024

Assessing AF2's ability to predict structural ensembles of proteins

Recent breakthroughs in protein structure prediction have enhanced the precision and speed at which protein configurations can be determined, setting new benchmarks for accuracy and efficiency in the field. However, the fundamental mechanisms of biological processes at a molecular level are often connected to conformational changes of proteins. Molecular dynamics (MD) simulations serve as a crucial tool for capturing the conformational space of proteins, providing valuable insights into their structural fluctuations. However, the scope of MD simulations is often limited by the accessible timescales and the computational resources available, posing challenges to comprehensively exploring protein behaviors. Recently emerging approaches have focused on expanding the capability of AlphaFold2 (AF2) to predict conformational substates of protein structures by manipulating the input multiple sequence alignment (MSA). These approaches operate under the assumption that the MSA also contains information about the heterogeneity of protein structures. Here, we benchmark the performance of various workflows that have adapted AF2 for ensemble prediction focusing on the subsampling of the MSA as implemented in ColabFold and compare the obtained structures with ensembles obtained from MD simulations and NMR. As test cases, we chose four proteins namely the bovine pancreatic inhibitor protein (BPTI), thrombin and two antigen binding fragments (antibody Fv and nanobody), for which reliable experimentally validated structural information (X-ray and/or NMR) was available. Thus, we provide an overview of the levels of performance and accessible timescales that can currently be achieved with machine learning (ML) based ensemble generation. In three out of the four test cases, we find structural variations fall within the predicted ensembles. Nevertheless, significant minima of the free energy surfaces remain undetected. This study highlights the possibilities and pitfalls when generating ensembles with AF2 and thus may guide the development of future tools while informing upon the results of currently available applications.

biophysics↗

bioRxiv2024

Mispacking of the F87 sidechain drives aggregation-promoting conformational fluctuations in the subunit interfaces of the transthyretin tetramer

Aberrant formation and deposition of human transthyretin (TTR) aggregates causes transthyretin amyloidosis. To initialize aggregation, transthyretin tetramers must first dissociate into monomers that partially unfold to promote entry into the aggregation pathway. The native TTR tetramer (T) is stabilized by docking of the F87 sidechain into an interfacial cavity enclosed by several hydrophobic residues including A120. We have previously shown that an alternative tetramer (T*) with mispacked F87 sidechains is more prone to dissociation and aggregation than the native T state. However, the molecular basis for the reduced stability in T* remains unclear. Here we report characterization of the A120L mutant, where steric hindrance is introduced into the F87 binding site. The X-ray structure of A120L shows that the F87 sidechain is displaced from its docking site across the subunit interface. In A120S, a naturally occurring pathogenic mutant that is less aggregation-prone than A120L, the F87 sidechain is correctly docked, as in the native TTR tetramer. Nevertheless, 19F-NMR aggregation assays show an elevated population of a monomeric aggregation intermediate in A120S relative to a control containing the native A120, due to accelerated tetramer dissociation and slowed monomer tetramerization. The mispacking of the F87 sidechain is associated with enhanced exchange dynamics for interfacial residues. At 298 K, the T* populations of various naturally occurring mutants fall between 4-7% ({Delta}G [~] 1.5- 1.9 kcal/mol), consistent with the free energy change expected for undocking and solvent exposure of one of the four F87 sidechains in the tetramer ({Delta}G [~] 1.6 kcal/mol). Our data provide a molecular-level picture of the likely universal F87 sidechain mispacking in tetrameric TTR that promotes interfacial conformational dynamics and increases aggregation propensity.

biophysics↗

bioRxiv2024

Characterization of an amyloidogenic intermediate of transthyretin by NMR relaxation dispersion

The aggregation pathway of transthyretin (TTR) proceeds through rate-limiting dissociation of the tetramer and partial misfolding of the monomers, which assemble into amyloid structures through a downhill polymerization mechanism. The structural features of the aggregation-prone monomeric intermediate are poorly understood. Characterization of amyloidogenic intermediates is challenging due to their propensity to aggregate at concentrations necessary for structural studies. NMR relaxation dispersion offers a unique opportunity to characterize these intermediates when they exchange on favorable timescales with NMR-visible ground states. To characterize the structural transitions associated with tetramer dissociation, we have analyzed ground-state chemical shift differences between the native tetramer and an engineered monomer in which the critical F87 side chain is replaced by glutamate. The secondary structure and overall fold of the F87E monomer is similar to that of the tetramer except for {beta}-strand H. This strand populates two conformations, where it is either docked on the protein core or is displaced from the edge of the {beta}-sheet formed by {beta}-strands D, A, G, and H (DAGH {beta}-sheet) and is dynamically disordered. Chemical shift differences derived from analysis of 1H/15N single, double and zero quantum relaxation dispersion data provide insights into the structure of a low-lying excited state that exchanges with the ground state of the F87E monomer at a rate of 3800 s-1. Disruption of the subunit interfaces of the TTR tetramer leads to destabilization of edge strands in both {beta}-sheets of the F87E monomer. Conformational fluctuations are propagated through the entire hydrogen bonding network of the DAGH {beta}-sheet, from the inner {beta}-strand H, which forms the strong dimer interface in the TTR tetramer, to outer strand D which is unfolded in TTR fibrils. Fluctuations are also propagated from the AB loop in the weak dimer interface to the EF helix, which undergoes structural remodeling in fibrils. The conformational fluctuations in both regions are enhanced at acidic pH where amyloid formation is most favorable. The relaxation dispersion data provide insights into the conformational dynamics of the amyloidogenic state of monomeric TTR that predispose it for structural remodeling and progression to amyloid fibrils.

biophysics↗

bioRxiv2024

Broadly inhibitory antibodies against severe malaria virulence proteins

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasites polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDR1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDR1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDR1 domains, representing five of the six subclasses of CIDR1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDR1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDR1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

immunology↗

bioRxiv2023

Probing the dissociation pathway of a kinetically labile transthyretin mutant

Aggregation of transthyretin (TTR) is associated with devastating TTR amyloid disease. Amyloidosis begins with dissociation of the native tetramer to form a monomeric intermediate that assembles into pathogenic aggregates. This process is accelerated in vitro at low pH, but the dissociation and reassembly of TTR at neutral pH remains poorly understood, due to the low population of intermediates. We use NMR studies with a highly sensitive 19F probe that allows deconvolution of relative populations of a destabilized A25T mutant at concentrations as low as 2 {micro}M. The A25T mutation, located at the weak dimer interface, perturbs both the weak and strong dimer interfaces. A tetramer-dimer-monomer (TDM) equilibrium model is proposed to account for concentration- and temperature-dependent population changes. All thermodynamic and kinetic parameters and activation energetics for dissociation of the native A25T tetramer, as well as a destabilized alternative tetramer (T*) with a mispacked F87 side chain, were extracted by vant Hoff and 19F NMR line-shape analysis. The conversion from T to T*, the slowest first-order kinetic step, shows anti-Arrhenius behavior. The 19F and methyl chemical shifts of probes close to the strong dimer interface in the dimer and T* species are degenerate, implicating interfacial perturbation as a common structural feature of these intermediate species. Molecular dynamics (MD) simulations further suggest more frequent F87 ring flipping on the nanoscale timescale in the A25T dimer than in the tetramer. Our integrated approach offers quantitative insights into the energy landscape of the dissociation pathway of TTR at neutral pH.

biophysics↗