Damien Maurin

High-throughput automated refolding screening of inclusion bodies

One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no “universal” refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high‐throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96‐well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large‐scale purification tests, and five gave crystals.

LppX is a lipoprotein required for the translocation of phthiocerol dimycocerosates to the surface of Mycobacterium tuberculosis

Cell envelope lipids play an important role in the pathogenicity of mycobacteria, but the mechanisms by which they are transported to the outer membrane of these prokaryotes are largely unknown. Here, we provide evidence that LppX is a lipoprotein required for the translocation of complex lipids, the phthiocerol dimycocerosates (DIM), to the outer membrane of Mycobacterium tuberculosis. Abolition of DIM transport following disruption of the lppX gene is accompanied by an important attenuation of the virulence of the tubercle bacillus. The crystal structure of LppX unveils an U‐shaped β‐half‐barrel dominated by a large hydrophobic cavity suitable to accommodate a single DIM molecule. LppX shares a similar fold with the periplasmic molecular chaperone LolA and the outer membrane lipoprotein LolB, which are involved in the localization of lipoproteins to the outer membrane of Gram‐negative bacteria. Based on the structure and although an indirect participation of LppX in DIM transport cannot yet be ruled out, we propose LppX to be the first characterized member of a family of structurally related lipoproteins that carry lipophilic molecules across the mycobacterial cell envelope.

A medium-throughput crystallization approach

The first results of a medium-scale structural genomics program clearly demonstrate the value of using a medium-throughput crystallization approach based on a two-step procedure: a large screening step employing robotics, followed by manual or automated optimization of the crystallization conditions. The structural genomics program was based on cloning in the Gateway vectors pDEST17, introducing a long 21-residue tail at the N-terminus. So far, this tail has not appeared to hamper crystallization. In ten months, 25 proteins were subjected to crystallization; 13 yielded crystals, of which ten led to usable data sets and five to structures. Furthermore, the results using a robot dispensing 50-200 nl drops indicate that smaller protein samples can be used for crystallization. These still partial results might indicate present and future directions for those who have to make crucial choices concerning their crystallization platform in structural genomics programs.

Lipolytic enzymes in Mycobacterium tuberculosis

Mycobacterium tuberculosis is a bacterial pathogen that can persist for decades in an infected patient without causing a disease. In vivo, the tubercle bacillus present in the lungs store triacylglycerols in inclusion bodies. The same process can be observed in vitro when the bacteria infect adipose tissues. Indeed, before entering in the dormant state, bacteria accumulate lipids originating from the host cell membrane degradation and from de novo synthesis. During the reactivation phase, these lipids are hydrolysed and the infection process occurs. The degradation of both extra and intracellular lipids can be directly related to the presence of lipolytic enzymes in mycobacteria, which have been ignored during a long period particularly due to the difficulties to obtain a high expression level of these enzymes in M. tuberculosis. The completion of the M. tuberculosis genome offered new opportunity to this kind of study. The aim of this review is to focus on the recent results obtained in the field of mycobacterium lipolytic enzymes and although no experimental proof has been shown in vivo, it is tempting to speculate that these enzymes could be involved in the virulence and pathogenicity processes.

Structure of the Tetramerization Domain of Measles Virus Phosphoprotein

ABSTRACT The atomic structure of the stable tetramerization domain of the measles virus phosphoprotein shows a tight four-stranded coiled coil. Although at first sight similar to the tetramerization domain of the Sendai virus phosphoprotein, which has a hydrophilic interface, the measles virus domain has kinked helices that have a strongly hydrophobic interface and it lacks the additional N-terminal three helical bundles linking the long helices.

Insights into the Mechanism by Which Interferon-γ Basic Amino Acid Clusters Mediate Protein Binding to Heparan Sulfate

The extensive functional repertoire of heparin and heparan sulfate, which relies on their ability to interact with a large number of proteins, has recently emerged. To understand the forces that drive such interactions the binding of heparin to interferon-γ (IFNγ), used as a model system, was investigated. NMR-based titration experiments demonstrated the involvement of two adjacent cationic domains (D1: KTGKRKR and D2: RGRR), both of which are present within the carboxy-terminal sequence of the cytokine. Kinetic analysis showed that these two domains contribute differently to the interaction: D1 is required to form a complex and constitutes the actual binding site, whereas D2, although unable to associate with heparin by itself, increased the association rate of the binding. These data are consistent with the view that D2, through nonspecific electrostatic forces, places the two molecules in favorable orientations for productive binding within the encounter complex. This mechanism was supported by electrostatic potential analysis and thermodynamic investigations. They showed that D1 association to heparin is driven by both favorable enthalpic and entropic contributions, as expected for a binding sequence, but that D2 gives rise to entropic penalty, which opposes binding in a thermodynamic sense. The binding mechanism described herein, by which the D2 domain kinetically drives the interaction, has important functional consequences and gives a structural framework to better understand how specific are the interactions between proteins and heparin.

Identification of Dynamic Modes in an Intrinsically Disordered Protein Using Temperature-Dependent NMR Relaxation

The dynamic modes and time scales sampled by intrinsically disordered proteins (IDPs) define their function. Nuclear magnetic resonance (NMR) spin relaxation is probably the most powerful tool for investigating these motions delivering site-specific descriptions of conformational fluctuations from throughout the molecule. Despite the abundance of experimental measurement of relaxation in IDPs, the physical origin of the measured relaxation rates remains poorly understood. Here we measure an extensive range of auto- and cross-correlated spin relaxation rates at multiple magnetic field strengths on the C-terminal domain of the nucleoprotein of Sendai virus, over a large range of temperatures (268-298 K), and combine these data to describe the dynamic behavior of this archetypal IDP. An Arrhenius-type relationship is used to simultaneously analyze up to 61 relaxation rates per amino acid over the entire temperature range, allowing the measurement of local activation energies along the chain, and the assignment of physically distinct dynamic modes. Fast (τ ≤ 50 ps) components report on librational motions, a dominant mode occurs on time scales around 1 ns, apparently reporting on backbone sampling within Ramachandran substates, while a slower component (5-25 ns) reports on segmental dynamics dominated by the chain-like nature of the protein. Extending the study to three protein constructs of different lengths (59, 81, and 124 amino acids) substantiates the assignment of these contributions. The analysis is shown to be remarkably robust, accurately predicting a broad range of relaxation data measured at different magnetic field strengths and temperatures. The ability to delineate intrinsic modes and time scales from NMR spin relaxation will improve our understanding of the behavior and function of IDPs, adding a new and essential dimension to the description of this biologically important and ubiquitous class of proteins.

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin alpha

Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Förster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin α and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin α. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.

Crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis

With the aim of elucidating the biological function of hypothetical proteins unique amongst the Actynomyces sub‐group of bacteria, we have solved the crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis at 1.8 Å resolution. Rv1155 is a homodimer both in the crystal structure and in solution and folds into two separate domains consisting of a six‐stranded anti‐parallel β‐barrel fold flanked by two α‐helices and a helix‐turn‐helix domain. Both domains contribute to the formation of two deep clefts at the dimer interface. The overall fold of Rv1155 strikingly resembles that of flavin mononucleotide‐binding protein and pyridoxamine 5′‐phosphate oxydase, but the architecture of the putative binding pocket is markedly different, consistent with the lack of color of Rv1155 and its inability to bind FMN. Rv1155 thus appears to belong to a group of proteins with stringent conservation of the binding cleft, having evolved towards a new binding function.

Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence

Abstract Measles virus RNA genomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleo‐proteins (N) that bind the entire genome. N‐RNA provides the template for replication and transcription by the viral polymerase and is a promising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs. Here, we demonstrate self‐organization of N into NC‐like particles in vitro upon addition of RNA, providing a simple and versatile tool for investigating assembly. Real‐time NMR and fluorescence spectroscopy reveals biphasic assembly kinetics. Remarkably, assembly depends strongly on the RNA‐sequence, with the genomic 5′ end and poly‐Adenine sequences assembling efficiently, while sequences such as poly‐Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process.