Loïc Salmon

Intrinsic disorder in measles virus nucleocapsids

The genome of measles virus is encapsidated by multiple copies of the nucleoprotein (N), forming helical nucleocapsids of molecular mass approaching 150 Megadalton. The intrinsically disordered C-terminal domain of N (NTAIL) is essential for transcription and replication of the virus via interaction with the phosphoprotein P of the viral polymerase complex. The molecular recognition element (MoRE) of NTAIL that binds P is situated 90 amino acids from the folded RNA-binding domain (NCORE) of N, raising questions about the functional role of this disordered chain. Here we report the first in situ structural characterization of NTAIL in the context of the entire N-RNA capsid. Using nuclear magnetic resonance spectroscopy, small angle scattering, and electron microscopy, we demonstrate that NTAIL is highly flexible in intact nucleocapsids and that the MoRE is in transient interaction with NCORE. We present a model in which the first 50 disordered amino acids of NTAIL are conformationally restricted as the chain escapes to the outside of the nucleocapsid via the interstitial space between successive NCORE helical turns. The model provides a structural framework for understanding the role of NTAIL in the initiation of viral transcription and replication, placing the flexible MoRE close to the viral RNA and, thus, positioning the polymerase complex in its functional environment.

Protein Conformational Flexibility from Structure-Free Analysis of NMR Dipolar Couplings: Quantitative and Absolute Determination of Backbone Motion in Ubiquitin†

A robust procedure for the determination of protein-backbone motions on time scales of pico- to milliseconds directly from residual dipolar couplings has been developed that requires no additional scaling relative to external references. The results for ubiquitin (blue in graph: experimental N-HN order parameters) correspond closely to the amplitude, nature, and distribution of motion found in a 400 ns molecular-dynamics trajectory of ubiquitin (red).

Mapping the Potential Energy Landscape of Intrinsically Disordered Proteins at Amino Acid Resolution

Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology. New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins. Although dependences of some NMR parameters such as chemical shifts (CSs) or residual dipolar couplings (RDCs) on structural propensity are known, so that sampling regimes are often inferred from experimental observation, there is currently no framework that allows for a statistical mapping of the available Ramachandran space of each amino acid in terms of conformational propensity. In this study we develop such an approach, combining highly efficient conformational sampling with ensemble selection to map the backbone conformational sampling of IDPs on a residue specific level. By systematically analyzing the ability of NMR data to map the conformational landscape of disordered proteins, we identify combinations of RDCs and CSs that can be used to raise conformational degeneracies inherent to different data types, and apply these approaches to characterize the conformational behavior of two intrinsically disordered proteins, the K18 domain from Tau protein and N(TAIL) from measles virus nucleoprotein. In both cases, we identify the enhanced populations of turn and helical regions in key regions of the proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling.

A general method for constructing atomic-resolution RNA ensembles using NMR residual dipolar couplings: the basis for interhelical motions revealed.

The ability to modulate alignment and measure multiple independent sets of NMR residual dipolar couplings (RDCs) has made it possible to characterize internal motions in proteins at atomic resolution and with time scale sensitivity ranging from picoseconds up to milliseconds. The application of such methods to the study of RNA dynamics, however, remains fundamentally limited by the inability to modulate alignment and by strong couplings between internal and overall motions that complicate the quantitative interpretation of RDCs. Here, we address this problem by showing that RNA alignment can be generally modulated, in a controlled manner, by variable elongation of A-form helices and that the information contained within the measured RDCs can be extracted even in the presence of strong couplings between motions and overall alignment via structure-based prediction of alignment. Using this approach, four RDC data sets, and a broad conformational pool obtained from a 8.2 μs molecular dynamics simulation, we successfully construct and validate an atomic resolution ensemble of human immunodeficiency virus type I transactivation response element RNA. This ensemble reveals local motions in and around the bulge involving changes in stacking and hydrogen-bonding interactions, which are undetectable by traditional spin relaxation and drive global changes in interhelical orientation. This new approach broadens the scope of using RDCs in characterizing the dynamics of nucleic acids.

Mapping the population of protein conformational energy sub-states from NMR dipolar couplings.

The precision with which X-ray crystallography and nuclear magnetic resonance (NMR) have provided structural models of biologically active and inactive conformations of countless proteins belies an easily overlooked dilemma. Proteins are inherently dynamic, exhibiting conformational freedom on timescales from picoseconds to seconds, implicating structural rearrangements that are essential for their biological function. Classical structural biology determines static models, that afford little insight into the underlying conformational equilibrium. The role that structural dynamics play in biological processes can only be understood by characterizing all thermally accessible protein conformations and their populations. NMR spectroscopy is uniquely sensitive to the presence of conformational dynamics in solution. Residual dipolar couplings (RDCs) measured in weakly aligned proteins, scalar couplings, and chemical shifts, probe motions occurring on timescales faster than 100 s of microseconds. These parameters therefore offer general tools to characterize protein motion on physiologically important timescales. A common approach to the dynamic interpretation of RDCs is to combine experimental restraint terms with a classical potential-energy force field to develop a conformational ensemble in agreement with experimental data. RDCs have also been exploited to characterize the conformational space sampled by the protein backbone either by fitting experimental data to determine angular excursions of internuclear bond vectors, or in comparison with different levels of accelerated molecular dynamics (AMD) to describe the most appropriate ensemble. Comparison of motions modeled using the Gaussian axial fluctuation (GAF) model, with ensembles derived from restraint-free AMD, demonstrated that such methods can provide a convergent description of protein motion.

Multi‐Timescale Conformational Dynamics of the SH3 Domain of CD2‐Associated Protein using NMR Spectroscopy and Accelerated Molecular Dynamics

A complete understanding of the relationship between biological activity and molecular conformation requires an understanding of the thermally accessible potential energy landscape. An extensive set of experimental NMR residual dipolar couplings (RDCs) has been used to determine the conformational behavior of CD2AP SH3C on multiple timescales, using the Gaussian Axial Fluctuation model, and comparison to restraint-free accelerated molecular dynamics simulation. These robust analyses provide a comprehensive description of conformational fluctuations on picosecond to millisecond timescales. While the β-sheets show negligible slow motions, larger amplitude slow dynamics are found in the n-SRC and RT loops that mediate physiological interactions.

Measuring similarity between dynamic ensembles of biomolecules.

We present a simple and general approach termed REsemble for quantifying population overlap and structural similarity between ensembles. This approach captures improvements in the quality of ensembles determined using increasing input experimental data—improvements that go undetected when conventional methods for comparing ensembles are used—and reveals unexpected similarities between RNA ensembles determined using NMR and molecular dynamics simulations.

Insights into domain-domain motions in proteins and RNA from solution NMR.

Conspectus Many multidomain proteins and ribonucleic acids consist of domains that autonomously fold and that are linked together by flexible junctions. This architectural design allows domains to sample a wide range of positions with respect to one another, yet do so in a way that retains structural specificity, since the number of sampled conformations remains extremely small compared to the total conformations that would be sampled if the domains were connected by an infinitely long linker. This “tuned” flexibility in interdomain conformation is in turn used in many biochemical processes. There is great interest in characterizing the dynamic properties of multidomain systems, and moving beyond conventional descriptions in terms of static structures, toward the characterization of population-weighted ensembles describing a distribution of many conformations sampled in solution. There is also great interest in understanding the design principles and underlying physical and chemical interactions that specify the nature of interdomain flexibility. NMR spectroscopy is one of the most powerful techniques for characterizing motions in complex biomolecules and has contributed greatly toward our basic understanding of dynamics in proteins and nucleic acids and its role in folding, recognition, and signaling. Here, we review methods that have been developed in our laboratories to address these challenges. Our approaches are based on the ability of one domain of the molecule to self-align in a magnetic field, or to dominate the overall orientation of the molecule, so that the conformational freedom of other domains can be assessed by their degree of alignment induced by the aligned part. In turn, this self-alignment ability can be intrinsic or can be caused by tagging appropriate constructs to the molecule of interest. In general, self-alignment is due to magnetic susceptibility anisotropy. Nucleic acids with elongated helices have this feature, as well as several paramagnetic metal centers that can be found in, or attached to, a protein domain.

HdeB Functions as an Acid-protective Chaperone in Bacteria

Background: Periplasmic chaperones HdeA and HdeB are involved in the acid stress response in Escherichia coli. Results: HdeB requires its folded and dimeric state to protect E. coli from protein aggregation at pH 4. Conclusion: HdeA and HdeB use different mechanisms to prevent periplasmic protein aggregation, allowing them to function over a broad pH range. Significance: This study furthers the understanding of how enteric bacteria counteract acid stress. Enteric bacteria such as Escherichia coli utilize various acid response systems to counteract the acidic environment of the mammalian stomach. To protect their periplasmic proteome against rapid acid-mediated damage, bacteria contain the acid-activated periplasmic chaperones HdeA and HdeB. Activation of HdeA at pH 2 was shown to correlate with its acid-induced dissociation into partially unfolded monomers. In contrast, HdeB, which has high structural similarities to HdeA, shows negligible chaperone activity at pH 2 and only modest chaperone activity at pH 3. These results raised intriguing questions concerning the physiological role of HdeB in bacteria, its activation mechanism, and the structural requirements for its function as a molecular chaperone. In this study, we conducted structural and biochemical studies that revealed that HdeB indeed works as an effective molecular chaperone. However, in contrast to HdeA, whose chaperone function is optimal at pH 2, the chaperone function of HdeB is optimal at pH 4, at which HdeB is still fully dimeric and largely folded. NMR, analytical ultracentrifugation, and fluorescence studies suggest that the highly dynamic nature of HdeB at pH 4 alleviates the need for monomerization and partial unfolding. Once activated, HdeB binds various unfolding client proteins, prevents their aggregation, and supports their refolding upon subsequent neutralization. Overexpression of HdeA promotes bacterial survival at pH 2 and 3, whereas overexpression of HdeB positively affects bacterial growth at pH 4. These studies demonstrate how two structurally homologous proteins with seemingly identical in vivo functions have evolved to provide bacteria with the means for surviving a range of acidic protein-unfolding conditions.

Visualizing chaperone-assisted protein folding

Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone–substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperone Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.