Small-Angle X-ray Scattering Study of Changes in the Quaternary Structure of Nucleotide-Regulated Pyrophosphatase from Desulfitobacterium hafniense upon Ligand Binding in Solution

The regulation of nucleotide-regulated inorganic pyrophosphatases at a molecular level has been extensively studied in order to establish the mechanism of signal transduction between the active and regulatory sites of these enzymes. However, this issue cannot be ultimately addressed because of the lack of reliable structural data on the full-length protein and its interactions with ligands. The low-resolution structure of nucleotide-regulated pyrophosphatase from Desulfitobacterium hafniense was determined for the first time by small-angle X-ray scattering. The structural changes in the full-length enzyme upon binding of adenosine monophosphate and diadenosine tetraphosphate were revealed. In dilute solutions the protein was found to exist as a stable homotetramer, the structure of which depends on the nature of the bound ligand. The structural data are important for an understanding of the molecular basis for regulation of this family of enzymes

Effect of Buffer Composition on Conformational Flexibility of N-Terminal Fragments of Dps and the Nature of Interactions with DNA. Small-Angle X-Ray Scattering Study

The DNA-binding protein Dps plays a key role in the formation of Dps–DNA crystalline arrays in living bacterial cells, which allows bacteria to survive under stress conditions and under the influence of various adverse factors. Such genome-protective mechanisms can lead to the emergence of bacterial resistance to antibiotics and other drugs. Elucidation of the fundamental biochemical, genetic, and structural basis of the resistance is of primary importance for the development of strategies for combating and preventing bacterial resistance, as well as the elaboration of innovative therapeutic approaches. Conformational characteristics of Dps and its N-terminal fragments responsible for the nature of interactions of this protein with DNA in solution were studied by small-angle scattering

Formation of High-Order Structures in Solution by CBS-Pyrophosphatase from D. hafniense

To solve the question about the oligomeric state of wild-type CBS-pyrophosphatase (CBS-PPase) from D. hafniense, this enzyme has been studied using two independent structural methods: small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryoTEM). The formation of stable high-order structures (large helical associates) in a concentrated protein solution has been observed for the first time. It is also shown for the first time that the formation of these structures is a reversible process and the protein passes to the tetramer form (in which it usually exists in diluted solutions) at ligand attachment. The obtained results are important for understanding the functional features of CBS-PPase (in particular, gaining insight into the pathogenesis of some diseases)

Small-Angle X-ray Scattering Study of Macrophage Migration Inhibitory Factor Complexed with Albumin

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, which plays a pivotal role in the regulation of immune response. Hence, the search for new inhibitors of MIF tautomerase activity has attracted great attention. This protein is known to serve as a superligand, by involving in protein–protein interactions that are poorly studied. Macrophage migration inhibitory factor was prepared in complex with albumin, and its solution structure was studied. Difficulties encountered in performing this research were due to the fact that the sample was a mixture of the MIF–albumin complex and the individual proteins. The interaction was found to be weak and unstable. Three most probable models of the MIF–albumin complex were obtained using small-angle X-ray scattering and molecular docking simulation, and one of these models was shown to be preferable. One albumin molecule binds to the MIF trimer in the active-site region of the protein

Small-angle X-Ray analysis of macromolecular structure: the structure of protein NS2 (NEP) in solution

A complex structural analysis of nuclear export protein NS2 (NEP) of influenza virus A has been performed using bioinformatics predictive methods and small-angle X-ray scattering data. The behavior of NEP molecules in a solution (their aggregation, oligomerization, and dissociation, depending on the buffer composition) has been investigated. It was shown that stable associates are formed even in a conventional aqueous salt solution at physiological рН value. For the first time we have managed to get NEP dimers in solution, to analyze their structure, and to compare the models obtained using the method of the molecular tectonics with the spatial protein structure predicted by us using the bioinformatics methods. The results of the study provide a new insight into the structural features of nuclear export protein NS2 (NEP) of the influenza virus A, which is very important for viral infection development

Quasi-Atomistic Approach to Modeling of Liposomes

Small-angle X-ray scattering is an important structural tool for studying biological membranes; however, interpretation of scattering data remains a challenging problem. In most cases, analysis makes it possible to determine some structural parameters and the electron density profile of lipid bilayers, but no methods providing more detailed information (e.g., about the structural organization of vesicles) have been proposed yet. An approach making it possible to determine the main integral characteristics of liposomes using small-angle scattering is presented in this study. Within this approach a quasi-atomic model of liposome is built from individual lipid molecules, which form a sphere or a hollow ellipsoid. The method has been implemented in a computer program, verified on experimental small-angle X-ray scattering data, and proposed to analyze the structure of lipid vesicles and their interactions with proteins

Solution Structure, Self-Assembly, and Membrane Interactions of the Matrix Protein from Newcastle Disease Virus at Neutral and Acidic pH

Newcastle disease virus (NDV) is an enveloped paramyxovirus. The matrix protein of the virus (M-NDV) has an innate propensity to produce virus-like particles budding from the plasma membrane of the expressing cell without recruiting other viral proteins. The virus predominantly infects the host cell via fusion with the host plasma membrane or, alternatively, can use receptor-mediated endocytic pathways. The question arises as to what are the mechanisms supporting such diversity, especially concerning the assembling and membrane binding properties of the virus protein scaffold under both neutral and acidic pH conditions. Here, we suggest a novel method of M-NDV isolation in physiological ionic strength and employ a combination of small-angle X-ray scattering, atomic force microscopy with complementary structural techniques, and membrane interaction measurements to characterize the solution behavior/structure of the protein as well as its binding to lipid membranes at pH 4.0 and pH 7.0. We demonstrate that the minimal structural unit of the protein in solution is a dimer that spontaneously assembles in a neutral milieu into hollow helical oligomers by repeating the protein tetramers. Acidic pH conditions decrease the protein oligomerization state to the individual dimers, tetramers, and octamers without changing the density of the protein layer and lipid membrane affinity, thus indicating that the endocytic pathway is a possible facilitator of NDV entry into a host cell through enhanced scaffold disintegration

The Cytoplasmic Tail of Influenza A Virus Hemagglutinin and Membrane Lipid Composition Change the Mode of M1 Protein Association with the Lipid Bilayer

Influenza A virus envelope contains lipid molecules of the host cell and three integral viral proteins: major hemagglutinin, neuraminidase, and minor M2 protein. Membrane-associated M1 matrix protein is thought to interact with the lipid bilayer and cytoplasmic domains of integral viral proteins to form infectious virus progeny. We used small-angle X-ray scattering (SAXS) and complementary techniques to analyze the interactions of different components of the viral envelope with M1 matrix protein. Small unilamellar liposomes composed of various mixtures of synthetic or “native” lipids extracted from Influenza A/Puerto Rico/8/34 (H1N1) virions as well as proteoliposomes built from the viral lipids and anchored peptides of integral viral proteins (mainly, hemagglutinin) were incubated with isolated M1 and measured using SAXS. The results imply that M1 interaction with phosphatidylserine leads to condensation of the lipid in the protein-contacting monolayer, thus resulting in formation of lipid tubules. This effect vanishes in the presence of the liquid-ordered (raft-forming) constituents (sphingomyelin and cholesterol) regardless of their proportion in the lipid bilayer. We also detected a specific role of the hemagglutinin anchoring peptides in ordering of viral lipid membrane into the raft-like one. These peptides stimulate the oligomerization of M1 on the membrane to form a viral scaffold for subsequent budding of the virion from the plasma membrane of the infected cell

Influenza virus Matrix Protein M1 preserves its conformation with pH, changing multimerization state at the priming stage due to electrostatics

Influenza A virus matrix protein M1 plays an essential role in the virus lifecycle, but its functional and structural properties are not entirely defined. Here we employed small-angle X-ray scattering, atomic force microscopy and zeta-potential measurements to characterize the overall structure and association behavior of the full-length M1 at different pH conditions. We demonstrate that the protein consists of a globular N-terminal domain and a flexible C-terminal extension. The globular N-terminal domain of M1 monomers appears preserved in the range of pH from 4.0 to 6.8, while the C-terminal domain remains flexible and the tendency to form multimers changes dramatically. We found that the protein multimerization process is reversible, whereby the binding between M1 molecules starts to break around pH 6. A predicted electrostatic model of M1 self-assembly at different pH revealed a good agreement with zeta-potential measurements, allowing one to assess the role of M1 domains in M1-M1 and M1-lipid interactions. Together with the protein sequence analysis, these results provide insights into the mechanism of M1 scaffold formation and the major role of the flexible and disordered C-terminal domain in this process