Structures of biomolecular complexes by combination of NMR and cryoEM methods

CryoEM is presently providing structures of biocomplexes considered intractable to analysis by other structural techniques. NMR is playing an important role in delivering structural information on dynamics events and conformational heterogeneity. Impressive results were obtained by combining cryoEM and either liquid- or solid-state NMR, revealing the structures of cellular machines, filaments and amyloid fibrils. NMR solution structures of proteins and nucleic acids were fitted, together with crystallographic structures, into cryoEM maps of large complexes, to decipher their assembly mechanisms and describe their functional dynamics. Modelling based on solid-state NMR and cryoEM data provided 3D structure of filaments and fibrils. These NMR approaches validated, but also corrected, atomic models built de novo in cryoEM maps, and provided new structural data on flexible or structurally heterogeneous systems. Combination of cryoEM and NMR became an established hybrid approach in structural biology that significantly contributes to our understanding of functional mechanisms in supramolecular assemblies

Signatures of Le´vy flights with annealed disorder

We present theoretical and experimental results of Le´vy flights of light originating from a random walk of photons in a hot atomic vapor. In contrast to systems with quenched disorder, this system does not present any correlations between the position and the step length of the random walk. In an analytical model based on micro- scopic first principles including Doppler broadening we find anomalous Le´vy-type superdiffusion corresponding to a single-step size distribution P (x) ∝ x−(1+α), with α ≈ 1. We show that this step size distribution leads to a violation of Ohm’s law [Tdiff ∝ L−α/2 ⵬= L−1], as expected for a Le´vy walk of independent steps. Furthermore, the spatial profile of the transmitted light develops power-law tails [Tdiff(r) ∝ r−3−α]. In an experiment using a slab geometry with hot Rb vapor, we measured the total diffuse transmission Tdiff and the spatial profile of the transmitted light Tdiff(r). We obtained the microscopic Le´vy parameter α under macroscopic multiple scattering conditions paving the way to investigation of Le´vy flights in different atomic physics and astrophysics systems.We thank Dominique Delande for fruitful discussions and we acknowledge funding for N.M. and Q.B. by the french Direction Generale de l'Armement. R.P acknowledges the support of LABEX WIFI (Laboratory of Excellence ANR-10-LABX-24) within the French Program "Investments for the Future" under reference ANR-10-IDEX-0001-02 PSL*. E.J.N. and R.K. acknowledge the FCT/CNRS exchange program (441.00 CNRS)

Towards third generation matrix metalloproteinase inhibitors for cancer therapy

The failure of matrix metalloproteinase (MMP) inhibitor drug clinical trials in cancer was partly due to the inadvertent inhibition of MMP antitargets that counterbalanced the benefits of MMP target inhibition. We explore how MMP inhibitor drugs might be developed to achieve potent selectivity for validated MMP targets yet therapeutically spare MMP antitargets that are critical in host protection

Accounting for conformational variability in NMR structure of cyclopeptides: Ensemble averaging of interproton distance and coupling constant restraints

The application of an ensemble-averaging (EA) protocol to highlight conformational variability and to determine the interconverting conformations in NMR structure of cyclopeptides is described. Most of the NMR-based conformational studies of cyclopeptides reported in the literature rely on protocols that basically assume the existence of a single structure. This is sometimes referred as the one NOE (or ROE)/one distance hypothesis. In contrast, the EA protocol used in this work relies on a model that explicitly takes into account the averaging in NMR data and tests the significancy of the results which is very often disregarded in structure determination by NMR. This EA method was applied to the conformational analysis of the peptide cyclo(Gly-Pro-Phe-Gly-Pro-Nle) in DMSO by NMR. Qualitative analysis of the ROEs observed for this peptide indicates that it adopts the well-known double reverse turn structure. However, certain interproton distances derived from a set of ROESY experiments, as well as some coupling constants, are not compatible with the existence of a unique conformation but reflect the presence of several conformers in fast exchange on the NMR time scale. Therefore, structures consistent as ensemble with the NMR-derived restraints were determined using a restrained molecular-dynamics-based ensemble- averaging protocol which explicitly takes multiconformers into account and treats the restraints as ensemble-averaged quantities. The NMR-derived data used as input restraints in this EA protocol include the distance restraints (DR), the homonuclear coupling constants (J), and a large set of unambiguous antidistance restraints (ADR) that are generally disregarded in conformational analysis of cyclopeptides. The number of interconverting conformers was determined from the significance of the fit of the DR and ADR using the complete cross-validation method. The results shows that pairs of conformers give a satisfactory and significant fit of all NMR data. The conformational analysis of the interconverting partners reveals that the hexapeptide cyclo(Gly-Pro-Phe-Gly-Pro-Nle) exists in solution either as a βVIII-βII/iγ-βI or a βII-βII/βI-β-I equilibrium

Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild-type angiotensin-converting enzyme

Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild-type enzyme, we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE, whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results, changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N-domain and C-domain substrate selectivity data derived only from human ACE. © 2006 The Authors

Three-Dimensional Structure of Cyclohexapeptides Containing a Phosphinic Bond in Aqueous Solution: A Template for Zinc Metalloprotease Inhibitors. A NMR and Restrained Molecular Dynamics Study

The 3D structures of two phosphinic cyclic hexapeptide inhibitors of bacterial collagenase, cyclo-(Gly1-Pro2-Phe3ψ[PO2-CH2]Gly4-Pro5-Nle6) (compound I) and cyclo(Gly1-Pro2-D-Phe3ψ [PO2-CH2]-Gly4-Pro5-Nle6) (compound II), in aqueous solution, as derived from NMR spectroscopy and molecular dynamics simulations, are described. The general structures of these cyclic hexapeptides closely resemble the “canonic” two-reverse-turn structure, with the proline occupying the (i + 1) position of the turns and the glycine the connecting positions. The phosphinic bond is located between the (i + 2) and (i + 3) positions of one of these turns. However, a striking feature of the backbone structure of these peptides is the presence of double type VIII-turns in compound I, and in compound II of type VIII- and tentatively named type IX-turns. The comparison of the 3D structures of these two cyclic hexapeptides shows that the stereochemistry of the phenylalanylphosphinyl residue influences not only the local conformation but also the global topology of the peptide macrocycle. The differences in the 3D structure of these compounds are discussed in relation to their inhibitory potencies and with the view of using these constrained cyclic peptides as a scaffold for the development of rigid metalloproteases inhibitors. © 1995, American Chemical Society. All rights reserved

Structural determinants of RXPA380, a potent and highly selective inhibitor of the angiotensin-converting enzyme C-domain

RXPA380 (Cbz-PhePsi[PO2CH]Pro-Trp-OH) was reported recently as the first highly selective inhibitor of the C-domain of somatic angiotensin-converting enzyme (ACE), able to differentiate the two active sites of somatic ACE by a selectivity factor of more than 3 orders of magnitude. The contribution of each RXPA380 residue toward this remarkable selectivity was evaluated by studying several analogues of RXPA380. This analysis revealed that both pseudo-proline and tryptophan residues in the P-1’ and P-2’ positions of RXPA380 play a critical role in the selectivity of this inhibitor for the C-domain. This selectivity is not due to a preference of the C-domain for inhibitors bearing pseudo-proline and tryptophan residues, but rather reflects the poor accommodation of these inhibitor residues by the N-domain. A model of RXPA380 in complex with the ACE C-domain, based on the crystal structure of germinal ACE, highlights residues that may contribute to RXPA380 selectivity. From this model, striking differences between the N- and C-domains of ACE are observed for residues defining the S-2’ pocket. Of the twelve residues that surround the tryptophan side chain of RXPA380 in the C-domain, five are different in the N-domain. These differences in the S-2’ composition between the N- and C-domains are suggested to contribute to RXPA380 selectivity. The structural insights provided by this study should enhance understanding of the factors controlling the selectivity of the two domains of somatic ACE and allow the design of new selective ACE inhibitors

Crystal structure of full-length human collagenase 3 (MMP-13) with peptides in the active site defines exosites in the catalytic domain.

Matrix metalloproteinase (MMP)-13 is one of the mammalian collagenases that play key roles in tissue remodelling and repair and in progression of diseases such as cancer, arthritis, atherosclerosis, and aneurysm. For collagenase to cleave triple helical collagens, the triple helical structure has to be locally unwound before hydrolysis, but this process is not well understood. We report crystal structures of catalytically inactive full-length human MMP-13(E223A) in complex with peptides of 14-26 aa derived from the cleaved prodomain during activation. Peptides are bound to the active site of the enzyme by forming an extended β-strand with Glu(40) or Tyr(46) inserted into the S1' specificity pocket. The structure of the N-terminal part of the peptides is variable and interacts with different parts of the catalytic domain. Those areas are designated substrate-dependent exosites, in that they accommodate different peptide structures, whereas the precise positioning of the substrate backbone is maintained in the active site. These modes of peptide-MMP-13 interactions have led us to propose how triple helical collagen strands fit into the active site cleft of the collagenase

Evaluation of P1'-Diversified Phosphinic Peptides Leads to the Development of Highly Selective Inhibitors of MMP-11

Phosphinic peptides were previously reported to be potent inhibitors of several matrixins (MMPs). To identify more selective inhibitors of MMP-11, a matrixin overexpressed in breast cancer, a series of phosphinic pseudopeptides bearing a variety of P1'-side chains has been synthesized, by parallel diversification of a phosphinic template. The potencies of these compounds were evaluated against a set of seven MMPs (MMP-2, MMP-7, MP-8, MMP-9, MMP-11, MMP-13, and MMP-14). The chemical strategy applied led to the identification of several phosphinic inhibitors displaying high selectivity toward MMP-11. One of the most selective inhibitors of MMP-11 in this series, compound 22, exhibits a Ki value of 0.23 μM toward MMP-11, while its potency toward the other MMPs tested is 2 orders of magnitude lower. This remarkable selectivity may rely on interactions of the P1'-side chain atoms of these inhibitors with residues located at the entrance of the S1'-cavity of MMP-11. The design of inhibitors able to interact with residues located at the entrance of MMPs' S1'-cavity might represent an alternative strategy to identify selective inhibitors that will fully differentiate one MMP among the others

Cyclic Peptides with a Phosphinic Bond as Potent Inhibitors of a Zinc Bacterial Collagenase

A series of cyclic peptides containing a phosphinic bond were synthesized and evaluated as inhibitors of a zinc bacterial collagenase from Corynebaeterium rathaii. Among this series of pseudopeptides of different sizes of cycles, only two molecules Ia (cyclo[Gly-Pro-Pheψ(PO2CH2)Gly-Pro-Ahx]) and Va (cyclo[βAla-Pro-Phe (PO2CH2)Gly-Pro-Ahx]) were found to be rather potent inhibitors of this protease, with Ki values of 120 and 90 nM, respectively. Besides the influence of the peptide ring size, this study suggests that both the stereochemical and the conformational properties of the pseudophenylalanine residue in these cyclic peptides may determine their potency. Interestingly, the kinetic analysis for the binding of the cyclic peptide inhibitors Ia and Va to the collagenase, as compared to a linear parent compound, reveals that the lower potency of the cyclic peptides is mostly the consequence of a lower rate constant for association to the enzyme. To our knowledge, this is the first report on cyclic phosphinic peptides and on their activities as inhibitors of a zinc protease. © 1994, American Chemical Society. All rights reserved