Julien Orts

Solution NMR Studies of Recombinant Aβ(1–42): From the Presence of a Micellar Entity to Residual β-Sheet Structure in the Soluble Species

Amyloid‐β (Aβ) peptide is the major component found in senile plaques of Alzheimers disease patients. The 42‐residue fragment Aβ(1–42) is proposed to be one of the most pathogenic species therein. Here, the soluble Aβ(1–42) species were analyzed by various liquid‐state NMR methods. Transient formation of a micelle species was observed at the onset of the aggregation kinetics. This micelle is dissolved after approximately one day. Subsequent loss of this species and the formation of protofibrils are proposed to be the route of fibril formation. Consequently, the observed micelle species is suggested to be on an off‐pathway mechanism. Furthermore, characterization of the NMR‐observable soluble species shows that it is a random‐coil‐like entity with low propensities for four β‐strands. These β‐strands correlate with the β‐strand segments observed in Aβ fibrils. This finding indicates that the 3D structure of the fibrils might already be predisposed in the soluble species.

Relaxation Matrix Analysis of Spin Diffusion for the NMR Structure Calculation with eNOEs

NMR structure determination is usually based on distance restraints extracted semiquantitatively from cross peak volumes or intensities in NOESY spectra. The recent introduction of exact NOEs (eNOE) by Vogeli et al. opens an avenue for the ensemble-based structure determination of proteins on the basis of eNOE-derived quantitative distance restraints. We present an approach to extract eNOE from build-up curve intensities. For the determination of eNOEs, spin diffusion is a major source of errors. A full relaxation matrix analysis is used to calculate the spin diffusion contribution to the NOESY cross peaks of each individual spin pair of interest. A software program is written, which requires as input the peak intensities from the various NOESY spectra as well as a 3D structure of the protein. This structure can be either an X-ray structure or an NMR structure determined with the conventional approach. The outputs of the program are the eNOE rates, the autorelaxation rates, as well as graphs and quality factors from the individual NOE build-up curves for semiautomated analysis of the derived rates. The protocol is straightforward, and the program integrates well into the current structure calculation workflow.

NMR-Based Determination of the 3D Structure of the Ligand–Protein Interaction Site without Protein Resonance Assignment

Molecular replacement in X-ray crystallography is the prime method for establishing structure-activity relationships of pharmaceutically relevant molecules. Such an approach is not available for NMR. Here, we establish a comparable method, called NMR molecular replacement (NMR(2)). The method requires experimentally measured ligand intramolecular NOEs and ligand-protein intermolecular NOEs as well as a previously known receptor structure or model. Our findings demonstrate that NMR(2) may open a new avenue for the fast and robust determination of the interaction site of ligand-protein complexes at atomic resolution.

Towards a true protein movie: A perspective on the potential impact of the ensemble-based structure determination using exact NOEs

Confined by the Boltzmann distribution of the energies of the states, a multitude of structural states are inherent to biomolecules. For a detailed understanding of a proteins function, its entire structural landscape at atomic resolution and insight into the interconversion between all the structural states (i.e. dynamics) are required. Whereas dedicated trickery with NMR relaxation provides aspects of local dynamics, and 3D structure determination by NMR is well established, only recently have several attempts been made to formulate a more comprehensive description of the dynamics and the structural landscape of a protein. Here, a perspective is given on the use of exact NOEs (eNOEs) for the elucidation of structural ensembles of a protein describing the covered conformational space.

A Structural Ensemble for the Enzyme Cyclophilin Reveals an Orchestrated Mode of Action at Atomic Resolution

For enzyme activity, an exact structural and motional orchestration of the active site and its surroundings is believed to be key. In order to reveal such possible phenomena at atomic resolution on the basis of experimental evidence, an experimental restraint driven two-state ensemble of the prototypical enzyme cyclophilin was determined by using a recently introduced exact NOE approach. The ensemble description reveals the presence of an open and a closed state of cyclophilin, which is indicative of large-scale correlated motion. In the open state, the catalytic site is preorganized for catalysis, thus suggesting the mechanism of action to be conformational sampling, while the ligand-binding loop appears to act through an induced fit mechanism. This finding is supported by affinity measurements of a cyclophilin designed to be more open. Overall, more than 60-70 % of the side-chain conformations of cyclophilin appear to be correlated.

The experimental accuracy of the uni-directional exact NOE.

We have established protocols to calculate exact NOEs (eNOE) from NOE data. eNOEs lend unprecedented precision to the calculation of distance restraints used for structure calculation. Moreover, as eNOEs are averaged quantities over all conformations of a molecule, they may contain accessible information of the sampled conformational space. In practice, a prerequisite for an exact interpretation is the evaluation of both NOESY cross-peak buildups. For large molecular sizes, the fraction of NOEs which can only be obtained from one cross peak typically increases. Distance restraints derived from such NOEs must be used with a tolerance for errors associated with the broken symmetry of the individual magnetization transfer pathways. The correct choice of upper and lower limits is particularly important for multiple-state ensemble calculation, where too narrow tolerances may lead to incorrect spatial sampling. In order to dissect these pathways in heavy-atom resolved 3D NOESY experiments, we analyze 2D [(1)H, (1)H]-NOESY experiments, which are the fundamental building blocks of the former. In combination with an analysis of excitation and inversion profiles of pulses on heavy atoms and relaxation effects during HXQC elements, we derive a rule for the correct choice of upper and lower distance limits derived from such uni-directional NOEs. We show that normalization of the cross- to the diagonal-peak intensities of the spins of magnetization destination rather than origin leads to similar errors of the distance restraints. This opens up the prospect of extended collection of unidirectional eNOEs.

Fast NMR-Based Determination of the 3D Structure of the Binding Site of Protein–Ligand Complexes with Weak Affinity Binders

In early drug discovery approaches, screening hits are often weak affinity binders that are difficult to characterize in structural detail, particularly towards obtaining the 3D structure of protein-ligand complexes at atomic resolution. NMR is the outstanding technique to tackle such problems, yet suffers from a tedious structure calculation process. NMR2 was recently developed to alleviate the laborious element of routine NMR structure calculation procedures and provides the structural information at protein-ligand interaction sites orders of magnitude faster than standard procedures. The NMR2 method was extended to weak binders and applied to the oncoproteins HDM2 and MDMX. The structure of the MDMX-SJ212 complex is reported with a Kd of approximately 0.7 μm; the complex structure of HDM2 with the mm affinity ligand #845 exhibits a new scaffold.

Discrete Three-dimensional Representation of Macromolecular Motion from eNOE-based Ensemble Calculation

Three-dimensional structural data and description of dynamics are fundamental to infer and understand protein function. Structure determination by NMR follows well-established protocols while NMR relaxation phenomena provide insights into local molecular dynamics. However, methods to detect concerted motion were not pursued until very recently. Here, we present an ensemble-based structure determination protocol using ensemble-averaged distance restraints obtained from exact NOE (eNOE) rate constants. An application of our protocol to the model protein GB3 established an ensemble of structures that reveals correlated motion across the β-sheet and concerted motion between the backbone and side chains localized in the core. Furthermore, the data repudiate concerted conformational exchange between the β-sheet and the α-helix.

The Exact Nuclear Overhauser Enhancement: Recent Advances

Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distance restraints, such that the multi-state structure calculation can be applied to proteins of higher molecular weights. We then review the implications of the exact NOE to the protein dynamics and function of cyclophilin A and the WW domain of Pin1, and finally discuss our current research and future directions.

Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.