Joseph E. Curtis

HIV-1 Gag extension: conformational changes require simultaneous interaction with membrane and nucleic acid.

The retroviral Gag polyprotein mediates viral assembly. The Gag protein has been shown to interact with other Gag proteins, with the viral RNA, and with the cell membrane during the assembly process. Intrinsically disordered regions linking ordered domains make characterization of the protein structure difficult. Through small-angle scattering and molecular modeling, we have previously shown that monomeric human immunodeficiency virus type 1 (HIV-1) Gag protein in solution adopts compact conformations. However, cryo-electron microscopic analysis of immature virions shows that in these particles, HIV-1 Gag protein molecules are rod shaped. These differing results imply that large changes in Gag conformation are possible and may be required for viral formation. By recapitulating key interactions in the assembly process and characterizing the Gag protein using neutron scattering, we have identified interactions capable of reversibly extending the Gag protein. In addition, we demonstrate advanced applications of neutron reflectivity in resolving Gag conformations on a membrane. Several kinds of evidence show that basic residues found on the distal N- and C-terminal domains enable both ends of Gag to bind to either membranes or nucleic acid. These results, together with other published observations, suggest that simultaneous interactions of an HIV-1 Gag molecule with all three components (protein, nucleic acid, and membrane) are required for full extension of the protein.

Observation of Small Cluster Formation in Concentrated Monoclonal Antibody Solutions and Its Implications to Solution Viscosity

Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals. It is hypothesized that some concentrated mAb solutions exhibit formation of a solution phase consisting of reversibly self-associated aggregates (or reversible clusters), which is speculated to be responsible for their distinct solution properties. Here, we report direct observation of reversible clusters in concentrated solutions of mAbs using neutron spin echo. Specifically, a stable mAb solution is studied across a transition from dispersed monomers in dilute solution to clustered states at more concentrated conditions, where clusters of a preferred size are observed. Once mAb clusters have formed, their size, in contrast to that observed in typical globular protein solutions, is observed to remain nearly constant over a wide range of concentrations. Our results not only conclusively establish a clear relationship between the undesirable high viscosity of some mAb solutions and the formation of reversible clusters with extended open structures, but also directly observe self-assembled mAb protein clusters of preferred small finite size similar to that in micelle formation that dominate the properties of concentrated mAb solutions.

SASSIE: A program to study intrinsically disordered biological molecules and macromolecular ensembles using experimental scattering restraints

Abstract A program to construct ensembles of biomolecular structures that are consistent with experimental scattering data are described. Specifically, we generate an ensemble of biomolecular structures by varying sets of backbone dihedral angles that are then filtered using experimentally determined restraints to rapidly determine structures that have scattering profiles that are consistent with scattering data. We discuss an application of these tools to predict a set of structures for the HIV-1 Gag protein, an intrinsically disordered protein, that are consistent with small-angle neutron scattering experimental data. We have assembled these algorithms into a program called SASSIE for structure generation, visualization, and analysis of intrinsically disordered proteins and other macromolecular ensembles using neutron and X-ray scattering restraints. Program summary Program title: SASSIE Catalogue identifier: AEKL_v1_0 Program summary URL: http://cpc.cs.qub.ac.uk/summaries/AEKL_v1_0.html Program obtainable from: CPC Program Library, Queenʼs University, Belfast, N. Ireland Licensing provisions: GNU General Public License v3 No. of lines in distributed program, including test data, etc.: 3 991 624 No. of bytes in distributed program, including test data, etc.: 826 Distribution format: tar.gz Programming language: Python, C/C++, Fortran Computer: PC/Mac Operating system: 32- and 64-bit Linux (Ubuntu 10.04, Centos 5.6) and Mac OS X (10.6.6) RAM: 1 GB Classification: 3 External routines: Python 2.6.5, numpy 1.4.0, swig 1.3.40, scipy 0.8.0, Gnuplot-py-1.8, Tcl 8.5, Tk 8.5, Mac installation requires aquaterm 1.0 (or X window system) and Xcode 3 development tools. Nature of problem: Open source software to generate structures of disordered biological molecules that subsequently allow for the comparison of computational and experimental results is limiting the use of scattering resources. Solution method: Starting with an all atom model of a protein, for example, users can input regions to vary dihedral angles, ensembles of structures can be generated. Additionally, simple two-body rigid-body rotations are supported with and without disordered regions. Generated structures can then be used to calculate small-angle scattering profiles which can then be filtered against experimentally determined data. Filtered structures can be visualized individually or as an ensemble using density plots. In the modular and expandable program framework the user can easily access our subroutines and structural coordinates can be easily obtained for study using other computational physics methods. Additional comments: The distribution file for this program is over 159 Mbytes and therefore is not delivered directly when download or Email is requested. Instead an html file giving details of how the program can be obtained is sent. Running time: Varies depending on application. Typically 10 minutes to 24 hours depending on the number of generated structures. References: [1] M.S. Kent, J.K. Murton, S. Satija, B. Akgun, H. Nanda, J.E. Curtis, J. Majewski, J.R. Engen, C.R. Morgan, Neutron reflectivity study of the conformation of HIV nef bound to lipid membranes, Biophys. J. 99 (6) (2010) 1940–1948. [2] H. Nanda, S.A.K. Datta, F. Heinrich, M. Losche, A. Rein, S. Krueger, J.E. Curtis, Electrostatic interactions and binding orientation of HIV-1 matrix, studied by neutron reflectivity, Biophys. J. 99 (7) (2010) 2516–2524. [3] S.A.K. Datta, J.E. Curtis, W. Ratcli, P.K. Clark, R.M. Crist, J. Lebowitz, S. Krueger, A. Rein, Conformation of the HIV-1 Gag protein in solution, J. Mol. Biol. 365 (3) (2007) 812–824.

Structural model of an mRNA in complex with the bacterial chaperone Hfq

Significance Small noncoding RNAs optimize bacterial gene expression under stress and increase the virulence of many bacterial pathogens. The RNA-binding protein Hfq (host factor Q-beta phage) promotes base pairing between small RNAs and target mRNAs, but it is not known how Hfq brings the two RNAs together in the proper orientation. We used chemical footprinting, small-angle X-ray scattering, and molecular dynamics simulations to model the structure of Hfq bound to an mRNA in solution. The surprising result is that the mRNA wraps entirely around the Hfq protein, specifically contacting both surfaces. This destabilizes the mRNA structure around the small RNA target site, poising it to base pair with a complementary small RNA also bound to Hfq. The Sm-like protein Hfq (host factor Q-beta phage) facilitates regulation by bacterial small noncoding RNAs (sRNAs) in response to stress and other environmental signals. Here, we present a low-resolution model of Escherichia coli Hfq bound to the rpoS mRNA, a bacterial stress response gene that is targeted by three different sRNAs. Selective 2′-hydroxyl acylation and primer extension, small-angle X-ray scattering, and Monte Carlo molecular dynamics simulations show that the distal face and lateral rim of Hfq interact with three sites in the rpoS leader, folding the RNA into a compact tertiary structure. These interactions are needed for sRNA regulation of rpoS translation and position the sRNA target adjacent to an sRNA binding region on the proximal face of Hfq. Our results show how Hfq specifically distorts the structure of the rpoS mRNA to enable sRNA base pairing and translational control.

Electrostatic Interactions and Binding Orientation of HIV-1 Matrix Studied by Neutron Reflectivity

The N-terminal matrix (MA) domain of the HIV-1 Gag protein is responsible for binding to the plasma membrane of host cells during viral assembly. The putative membrane-binding interface of MA was previously mapped by means of mutagenesis and analysis of its trimeric crystal structure. However, the orientation of MA on membranes has not been directly determined by experimental measurements. We present neutron reflectivity measurements that resolve the one-dimensional scattering length density profile of MA bound to a biomimetic of the native viral membrane. A molecular refinement procedure was developed using atomic structures of MA to determine the orientation of the protein on the membrane. The orientation defines a lipid-binding interface consistent with previous mutagenesis results. The MA protein maintains this orientation without the presence of a myristate group, driven only by electrostatic interactions. Furthermore, MA is found to penetrate the membrane headgroup region peripherally such that only the side chains of specific Lys and Arg residues interact with the surface. The results suggest that electrostatic interactions are sufficient to favorably orient MA on viral membrane mimics. The spatial determination of the membrane-bound protein demonstrates the ability of neutron reflectivity to discern orientation and penetration under physiologically relevant conditions.

Small-angle neutron scattering study of a monoclonal antibody using free-energy constraints.

Monoclonal antibodies (mAbs) contain hinge-like regions that enable structural flexibility of globular domains that have a direct effect on biological function. A subclass of mAbs, IgG2, have several interchain disulfide bonds in the hinge region that could potentially limit structural flexibility of the globular domains and affect the overall configuration space available to the mAb. We have characterized human IgG2 mAb in solution via small-angle neutron scattering (SANS) and interpreted the scattering data using atomistic models. Molecular Monte Carlo combined with molecular dynamics simulations of a model mAb indicate that a wide range of structural configurations are plausible, spanning radius of gyration values from ∼39 to ∼55 Å. Structural ensembles and representative single structure solutions were derived by comparison of theoretical SANS profiles of mAb models to experimental SANS data. Additionally, molecular mechanical and solvation free-energy calculations were carried out on the ensemble of best-fitting mAb structures. The results of this study indicate that low-resolution techniques like small-angle scattering combined with atomistic molecular simulations with free-energy analysis may be helpful to determine the types of intramolecular interactions that influence function and could lead to deleterious changes to mAb structure. This methodology will be useful to analyze small-angle scattering data of many macromolecular systems.

Solution Conformations of Prototype Foamy Virus Integrase and Its Stable Synaptic Complex with U5 Viral DNA

Using small-angle X-ray and neutron scattering (SAXS/SANS), in combination with analytical centrifugation and light scattering, we have determined the solution properties of PFV IN alone and its synaptic complex with processed U5 viral DNA and related these properties to models derived from available crystal structures. PFV IN is a monomer in solution, and SAXS analysis indicates an ensemble of conformations that differ from that observed in the crystallographic DNA-bound state. Scattering data indicate that the PFV intasome adopts a shape in solution that is consistent with the tetrameric assembly inferred from crystallographic symmetry, and these properties are largely preserved in the presence of divalent ions and clinical strand transfer inhibitors. Using contrast variation methods, we have reconstructed the solution structure of the PFV intasome complex and have located the distal domains of IN that were unresolved by crystallography. These results provide important insights into the architecture of the retroviral intasome.

Pronounced Microheterogeneity in a Sorbitol-Water Mixture Observed through Variable Temperature Neutron Scattering

In this study, the structure of concentrated d-sorbitol-water mixtures is studied by wide- and small-angle neutron scattering (WANS and SANS) as a function of temperature. The mixtures are prepared using both deuterated and regular sorbitol and water at a molar fraction of sorbitol of 0.19 (equivalent to 70% by weight of regular sorbitol in water). Retention of an amorphous structure (i.e., absence of crystallinity) is confirmed for this system over the entire temperature range, 100-298 K. The glass transition temperature, Tg, is found from differential scanning calorimetry to be approximately 200 K. WANS data are analyzed using empirical potential structure refinement, to obtain the site-site radial distribution functions (RDFs) and coordination numbers. This analysis reveals the presence of nanoscaled water clusters surrounded by (and interacting with) sorbitol molecules. The water clusters appear more structured compared to bulk water and, especially at the lowest temperatures, resemble the structure of low-density amorphous ice (LDA). Upon cooling to 100 K the peaks in the water RDFs become markedly sharper, with increased coordination number, indicating enhanced local (nanometer-scale) ordering, with changes taking place both above and well below the Tg. On the mesoscopic (submicrometer) scale, although there are no changes between 298 and 213 K, cooling the sample to 100 K results in a significant increase in the SANS signal, which is indicative of pronounced inhomogeneities. This increase in the scattering is partly reversed during heating, although some hysteresis is observed. Furthermore, a power law analysis of the SANS data indicates the existence of domains with well-defined interfaces on the submicrometer length scale, probably as a result of the appearance and growth of microscopic voids in the glassy matrix. Because of the unusual combination of small and wide scattering data used here, the present results provide new physical insight into the structure of aqueous glasses over a broad temperature and length scale, leading to an improved understanding of the mechanisms of temperature- and water-induced (de)stabilization of various systems, including proteins, pharmaceuticals, and biological objects.

Atomistic modelling of scattering data in the Collaborative Computational Project for Small Angle Scattering (CCP-SAS)

The CCP-SAS project is currently developing software for the atomistic and coarse-grained molecular modelling of X-ray and neutron small-angle scattering data. Its computational framework is described, alongside applications in biology and soft matter.

Structural Features of Membrane-bound Glucocerebrosidase and α-Synuclein Probed by Neutron Reflectometry and Fluorescence Spectroscopy

Background: A specific interaction exists between α-synuclein and glucocerebrosidase on the lipid membrane, resulting in enzyme inhibition. Results: Binding glucocerebrosidase has a profound effect on α-synuclein, moving roughly half of its embedded helical region above the membrane plane. Conclusion: A model is proposed with structural insights into glucocerebrosidase inhibition by α-synuclein. Significance: α-Synuclein-glucocerebrosidase interaction provides a molecular connection between Parkinson and Gaucher diseases. Mutations in glucocerebrosidase (GCase), the enzyme deficient in Gaucher disease, are a common genetic risk factor for the development of Parkinson disease and related disorders, implicating the role of this lysosomal hydrolase in the disease etiology. A specific physical interaction exists between the Parkinson disease-related protein α-synuclein (α-syn) and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. Here, neutron reflectometry was employed as a first direct structural characterization of GCase and α-syn·GCase complex on a sparsely-tethered lipid bilayer, revealing the orientation of the membrane-bound GCase. GCase binds to and partially inserts into the bilayer with its active site most likely lying just above the membrane-water interface. The interaction was further characterized by intrinsic Trp fluorescence, circular dichroism, and surface plasmon resonance spectroscopy. Both Trp fluorescence and neutron reflectometry results suggest a rearrangement of loops surrounding the catalytic site, where they extend into the hydrocarbon chain region of the outer leaflet. Taking advantage of contrasting neutron scattering length densities, the use of deuterated α-syn versus protiated GCase showed a large change in the membrane-bound structure of α-syn in the complex. We propose a model of α-syn·GCase on the membrane, providing structural insights into inhibition of GCase by α-syn. The interaction displaces GCase away from the membrane, possibly impeding substrate access and perturbing the active site. GCase greatly alters membrane-bound α-syn, moving helical residues away from the bilayer, which could impact the degradation of α-syn in the lysosome where these two proteins interact.