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

Understanding Chemistry and Unique NMR Characters of Novel Amide and Ester Leflunomide Analogues

A series of diverse substituted 5-methyl-isoxazole-4-carboxylic acid amides, imide and esters in which the benzene ring is mono or disubstituted was prepared. Spectroscopic and conformational examination was investigated and a new insight involving steric interference and interesting downfield deviation due to additional diamagnetic anisotropic effect of the amidic carbonyl group and the methine protons in 2,6-diisopropyl-aryl derivative (2) as conformationaly restricted analogues Leflunomide was discussed. Individual substituent electronic effects through π resonance of p-substituents and most stable conformation of compound (2) are discussed

Structure and Formation of Z-DNA and Z-RNA

Despite structural differences between the right-handed conformations of A-RNA and B-DNA, both nucleic acids adopt very similar, left-handed Z-conformations. In contrast to their structural similarities and sequence preferences, RNA and DNA exhibit differences in their ability to adopt the Z-conformation regarding their hydration shells, the chemical modifications that promote the Z-conformation, and the structure of junctions connecting them to right-handed segments. In this review, we highlight the structural and chemical properties of both Z-DNA and Z-RNA and delve into the potential factors that contribute to both their similarities and differences. While Z-DNA has been extensively studied, there is a gap of knowledge when it comes to Z-RNA. Where such information is lacking, we try and extend the principles of Z-DNA stability and formation to Z-RNA, considering the inherent differences of the nucleic acids

High-resolution small RNA structures from exact nuclear Overhauser enhancement measurements without additional restraints

RNA not only translates the genetic code into proteins, but also carries out important cellular functions. Understanding such functions requires knowledge of the structure and dynamics at atomic resolution. Almost half of the published RNA structures have been solved by nuclear magnetic resonance (NMR). However, as a result of severe resonance overlap and low proton density, high-resolution RNA structures are rarely obtained from nuclear Overhauser enhancement (NOE) data alone. Instead, additional semi-empirical restraints and labor-intensive techniques are required for structural averages, while there are only a few experimentally derived ensembles representing dynamics. Here we show that our exact NOE (eNOE) based structure determination protocol is able to define a 14-mer UUCG tetraloop structure at high resolution without other restraints. Additionally, we use eNOEs to calculate a two-state structure, which samples its conformational space. The protocol may open an avenue to obtain high-resolution structures of small RNA of unprecedented accuracy with moderate experimental efforts

Efficient Stereospecific Hβ2/3 NMR Assignment Strategy for Mid-Size Proteins

We present a strategy for stereospecific NMR assignment of Hβ2 and Hβ3 protons in mid-size proteins (~150 residues). For such proteins, resonance overlap in standard experiments is severe, thereby preventing unambiguous assignment of a large fraction of β-methylenes. To alleviate this limitation, assignment experiments may be run in high static fields, where higher decoupling power is required. Three-bond Hα–Hβ J-couplings (3JHα–Hβ) are critical for stereospecific assignments of β-methylene protons, and for determining rotameric χ1 states. Therefore, we modified a pulse sequence designed to measure accurate 3JHα–Hβ couplings such that probe heating was reduced, while the decoupling performance was improved. To further increase the resolution, we applied non-uniform sampling (NUS) schemes in the indirect 1H and 13C dimensions. The approach was applied to two medium-sized proteins, odorant binding protein 22 (OBP22; 14.4 kDa) and Pin1 (18.2 kDa), at 900 MHz polarizing fields. The coupling values obtained from NUS and linear sampling were extremely well correlated. However, NUS decreased the overlap of Hβ2/3 protons, thus supplying a higher yield of extracted 3JHα-Hβ coupling values when compared with linear sampling. A similar effect could be achieved with linear prediction applied to the linearly sampled data prior to the Fourier transformation. Finally, we used 3JHα–Hβ couplings from Pin1 in combination with either conventional or exact nuclear Overhauser enhancement (eNOE) restraints to determine the stereospecific assignments of β-methylene protons. The use of eNOEs further increased the fraction of unambiguously assigned resonances when compared with procedures using conventional NOEs

Ligand-specific conformational change drives interdomain allostery in Pin1

Pin1 is a two-domain cell regulator that isomerizes peptidyl-prolines. The catalytic domain (PPIase) and the other ligand-binding domain (WW) sample extended and compact conformations. Ligand binding changes the equilibrium of the interdomain conformations, but the conformational changes that lead to the altered domain sampling were unknown. Prior evidence has supported an interdomain allosteric mechanism. We recently introduced a magnetic resonance-based protocol that allowed us to determine the coupling of intra- and interdomain structural sampling in apo Pin1. Here, we describe ligand-specific conformational changes that occur upon binding of pCDC25c and FFpSPR. pCDC25c binding doubles the population of the extended states compared to the virtually identical populations of the apo and FFpSPR-bound forms. pCDC25c binding to the WW domain triggers conformational changes to propagate via the interdomain interface to the catalytic site, while FFpSPR binding displaces a helix in the PPIase that leads to repositioning of the PPIase catalytic loop.ISSN:2041-172

Reconstruction of Coupled Intra- and Interdomain Protein Motion from Nuclear and Electron Magnetic Resonance

Proteins composed of multiple domains allow for structural heterogeneity and interdomain dynamics that may be vital for function. Intradomain structures and dynamics can influence interdomain conformations and vice versa. However, no established structure determination method is currently available that can probe the coupling of these motions. The protein Pin1 contains separate regulatory and catalytic domains that sample “extended” and “compact” states, and ligand binding changes this equilibrium. Ligand binding and interdomain distance have been shown to impact the activity of Pin1, suggesting interdomain allostery. In order to characterize the conformational equilibrium of Pin1, we describe a novel method to model the coupling between intra- and interdomain dynamics at atomic resolution using multistate ensembles. The method uses time-averaged nuclear magnetic resonance (NMR) restraints and double electron–electron resonance (DEER) data that resolve distance distributions. While the intradomain calculation is primarily driven by exact nuclear Overhauser enhancements (eNOEs), J couplings, and residual dipolar couplings (RDCs), the relative domain distribution is driven by paramagnetic relaxation enhancement (PREs), RDCs, interdomain NOEs, and DEER. Our data support a 70:30 population of the compact and extended states in apo Pin1. A multistate ensemble describes these conformations simultaneously, with distinct conformational differences located in the interdomain interface stabilizing the compact or extended states. We also describe correlated conformations between the catalytic site and interdomain interface that may explain allostery driven by interdomain contact.ISSN:0002-7863ISSN:1520-512

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

A β-Turn Motif in the Steroid Hormone Receptor’s Ligand-Binding Domains Interacts with the Peptidyl-prolyl Isomerase (PPIase) Catalytic Site of the Immunophilin FKBP52

International audienceThe immunophilin FKBP52 interacts with nuclear steroid hormone receptors. Studying the crystal structure of human estrogen receptor alpha (hER alpha) and using nuclear magnetic resonance, we show here that the short V(364)PGF(367) sequence, which is located within its ligand-binding domain and adopts a type II beta-turn conformation in the protein, binds the peptidyl-prolyl isomerase (PPIase or rotamase) FK1 domain of FKBP52. Interestingly, this turn motif displays strong similarities with the FKBP52 FK1 domain-binding moiety of macrolide immunomodulators such as rapamycin and GPI-1046, an immunophilin ligand with neuroprotective characteristics. An increase in the hydrophobicity of the residue preceding the proline and cyclization of the VPGF peptide strengthen its recognition by the FK1 domain of FKBP52. Replacement of the Pro residue with a dimethylproline also enhances this interaction. Our study not only contributes to a better understanding of how the interaction between the FK1 domain of FKBP52 and steroid hormone receptors most likely works but also opens new avenues for the synthesis of FKBP52 FK1 peptide ligands appropriate for the control of hormone-dependent physiological mechanisms or of the functioning of the Tau protein. Indeed, it has been shown that FKBP52 is involved in the intraneuronal dynamics of the Tau protein