Site‐Resolved Backbone and Side‐Chain Intermediate Dynamics in a Carbohydrate‐Binding Module Protein Studied by Magic‐Angle Spinning NMR Spectroscopy

Magic‐angle spinning solid‐state NMR spectroscopy has been applied to study the dynamics of CBM3b–Cbh9A from Clostridium thermocellum (ctCBM3b), a cellulose binding module protein. This 146‐residue protein has a nine‐stranded β‐sandwich fold, in which 35 % of the residues are in the β‐sheet and the remainder are composed of loops and turns. Dynamically averaged ¹H‐¹³C dipolar coupling order parameters were extracted in a site‐specific manner by using a pseudo‐three‐dimensional constant‐time recoupled separated‐local‐field experiment (dipolar‐chemical shift correlation experiment; DIPSHIFT). The backbone‐Cα and Cβ order parameters indicate that the majority of the protein, including turns, is rigid despite having a high content of loops; this suggests that restricted motions of the turns stabilize the loops and create a rigid structure. Water molecules, located in the crystalline interface between protein units, induce an increased dynamics of the interface residues thereby lubricating crystal water‐mediated contacts, whereas other crystal contacts remain rigid.     

Natural Compounds in Cancer Prevention: Effects of Coffee Extracts and Their Main Polyphenolic Component, 5‐O‐Caffeoylquinic Acid,on Oncogenic Ras Proteins

Recent epidemiological studies have demonstrated that the consumption of healthy foods that are particularly rich in polyphenols might reduce the incidence of cancer and neurodegenerative diseases. In particular, chlorogenic acids (CGAs) occur ubiquitously in food and represent the most abundant polyphenols in the human diet. A number of beneficial biological effects of CGAs, such as anti‐inflammatory activity, anti‐carcinogenic activity, and protection against neurodegenerative diseases, have been reported. However, the molecular mechanisms at the base of these biological activities have not yet been investigated in depth. By combining NMR spectroscopy, molecular docking, surface plasmon resonance and ex vivo assays of the Ras‐dependent breast cancer cell line MDA‐MB‐231, we contribute to the elucidation of the molecular basis of the activity of CGAs and natural extracts from green and roasted coffee beans as chemoprotective dietary supplements.     

Conformational Dynamics of apo‐GlnBP Revealed by Experimental and Computational Analysis

The glutamine binding protein (GlnBP) binds l ‐glutamine and cooperates with its cognate transporters during glutamine uptake. Crystal structure analysis has revealed an open and a closed conformation for apo‐ and holo‐GlnBP, respectively. However, the detailed conformational dynamics have remained unclear. Herein, we combined NMR spectroscopy, MD simulations, and single‐molecule FRET techniques to decipher the conformational dynamics of apo‐GlnBP. The NMR residual dipolar couplings of apo‐GlnBP were in good agreement with a MD‐derived structure ensemble consisting of four metastable states. The open and closed conformations are the two major states. This four‐state model was further validated by smFRET experiments and suggests the conformational selection mechanism in ligand recognition of GlnBP.     

Site‐Specific Investigation of the Steady‐State Kinetics and Dynamics of the Multistep Binding of Bile Acid Molecules to a Lipid Carrier Protein

The investigation of multi‐site ligand–protein binding and multi‐step mechanisms is highly demanding. In this work, advanced NMR methodologies such as 2D ¹H–¹⁵N line‐shape analysis, which allows a reliable investigation of ligand binding occurring on micro‐ to millisecond timescales, have been extended to model a two‐step binding mechanism. The molecular recognition and complex uptake mechanism of two bile salt molecules by lipid carriers is an interesting example that shows that protein dynamics has the potential to modulate the macromolecule–ligand encounter. Kinetic analysis supports a conformational selection model as the initial recognition process in which the dynamics observed in the apo form is essential for ligand uptake, leading to conformations with improved access to the binding cavity. Subsequent multi‐step events could be modelled, for several residues, with a two‐step binding mechanism. The protein in the ligand‐bound state still exhibits a conformational rearrangement that occurs on a very slow timescale, as observed for other proteins of the family. A global mechanism suggesting how bile acids access the macromolecular cavity is thus proposed.     

NMR Structure of a Triangulenium‐Based Long‐Lived Fluorescence Probe Bound to a G‐Quadruplex

An NMR structural study of the interaction between a small‐molecule optical probe (DAOTA‐M2) and a G‐quadruplex from the promoter region of the c‐myc oncogene revealed that they interact at 1:2 binding stoichiometry. NMR‐restrained structural calculations show that binding of DAOTA‐M2 occurs mainly through π–π stacking between the polyaromatic core of the ligand and guanine residues of the outer G‐quartets. Interestingly, the binding affinities of DAOTA‐M2 differ by a factor of two for the outer G‐quartets of the unimolecular parallel G‐quadruplex under study. Unrestrained MD calculations indicate that DAOTA‐M2 displays significant dynamic behavior when stacked on a G‐quartet plane. These studies provide molecular guidelines for the design of triangulenium derivatives that can be used as optical probes for G‐quadruplexes.     

Two Distinct Structures of Membrane‐Associated Homodimers of GTP‐ and GDP‐Bound KRAS4B Revealed by Paramagnetic Relaxation Enhancement

KRAS homo‐dimerization has been implicated in the activation of RAF kinases, however, the mechanism and structural basis remain elusive. We developed a system to study KRAS dimerization on nanodiscs using paramagnetic relaxation enhancement (PRE) NMR spectroscopy, and determined distinct structures of membrane‐anchored KRAS dimers in the active GTP‐ and inactive GDP‐loaded states. Both dimerize through an α4–α5 interface, but the relative orientation of the protomers and their contacts differ substantially. Dimerization of KRAS‐GTP, stabilized by electrostatic interactions between R135 and E168, favors an orientation on the membrane that promotes accessibility of the effector‐binding site. Remarkably, “cross”‐dimerization between GTP‐ and GDP‐bound KRAS molecules is unfavorable. These models provide a platform to elucidate the structural basis of RAF activation by RAS and to develop inhibitors that can disrupt the KRAS dimerization. The methodology is applicable to many other farnesylated small GTPases.     

Mannose‐Binding Geometry of Pradimicin A

Pradimicins (PRMs) and benanomicins are the only family of non‐peptidic natural products with lectin‐like properties, that is, they recognize D ‐mannopyranoside (Man) in the presence of Ca²⁺ ions. Coupled with their unique Man binding ability, they exhibit antifungal and anti‐HIV activities through binding to Man‐containing glycans of pathogens. Notwithstanding the great potential of PRMs as the lectin mimics and therapeutic leads, their molecular basis of Man recognition has yet to be established. Their aggregate‐forming propensity has impeded conventional interaction analysis in solution, and the analytical difficulty is exacerbated by the existence of two Man binding sites in PRMs. In this work, we investigated the geometry of the primary Man binding of PRM‐A, an original member of PRMs, by the recently developed analytical strategy using the solid aggregate composed of the 1:1 complex of PRM‐A and Man. Evaluation of intermolecular distances by solid‐state NMR spectroscopy revealed that the C2–C4 region of Man is in close contact with the primary binding site of PRM‐A, while the C1 and C6 positions of Man are relatively distant. The binding geometry was further validated by co‐precipitation experiments using deoxy‐Man derivatives, leading to the proposal that PRM‐A binds not only to terminal Man residues at the non‐reducing end of glycans, but also to internal 6‐substituted Man residues. The present study provides new insights into the molecular basis of Man recognition and glycan specificity of PRM‐A.     

Extending the Lifetime of Native GTP‐Bound Ras for Site‐Resolved NMR Measurements: Quantifying the Allosteric Dynamics

Characterization of native GTP‐bound Ras is important for an appreciation of its cellular signaling and for the design of inhibitors, which however has been depressed by its intrinsic instability. Herein, an effective approach for extending the lifetime of Ras?GTP samples by exploiting the active role of Son of Sevenless (Sos) is demonstrated that sustains the activated state of Ras. This approach, combined with a postprocessing method that suppresses residual Ras?GDP signals, is applied to the site‐resolved NMR measurement of the allosteric dynamics of Ras?GTP. The observed network of concerted motions well covers the recently identified allosteric inhibitor‐binding pockets, but the motions are more confined than those of Ras?GppNHp, advocating the use of native GTP for development of allosteric inhibitors. The Sos‐based approach is anticipated to generally facilitate experiments on active Ras when native GTP is preferred.     

Kinetics of the Antibody Recognition Site in the Third IgG‐Binding Domain of Protein G

Protein dynamics occurring on a wide range of timescales play a crucial role in governing protein function. Particularly, motions between the globular rotational correlation time ( ) and 40 μs (supra‐ window), strongly influence molecular recognition. This supra‐ window was previously hidden, owing to a lack of experimental methods. Recently, we have developed a high‐power relaxation dispersion (RD) experiment for measuring kinetics as fast as 4 μs. For the first time, this method, performed under super‐cooled conditions, enabled us to detect a global motion in the first β‐turn of the third IgG‐binding domain of protein G (GB3), which was extrapolated to 371±115 ns at 310 K. Furthermore, the same residues show the plasticity in the model‐free residual dipolar coupling (RDC) order parameters and in an ensemble encoding the supra‐ dynamics. This β‐turn is involved in antibody binding, exhibiting the potential link of the observed supra‐ motion with molecular recognition.     

A Non‐damaging Method to Analyze the Configuration and Dynamics of Nitrotyrosines in Proteins

Often, deregulation of protein activity and turnover by tyrosine nitration drives cells toward pathogenesis. Hence, understanding how the nitration of a protein affects both its function and stability is of outstanding interest. Nowadays, most of the in vitro analyses of nitrated proteins rely on chemical treatment of native proteins with an excess of a chemical reagent. One such reagent, peroxynitrite, stands out for its biological relevance. However, given the excess of the nitrating reagent, the resulting in vitro modification could differ from the physiological nitration. Here, we determine unequivocally the configuration of distinct nitrated‐tyrosine rings in single‐tyrosine mutants of cytochrome c. We aimed to confirm the nitration position by a non‐destructive method. Thus, we have resorted to ¹H‐¹⁵N heteronuclear single quantum coherence(HSQC) spectra to identify the ³J(N? H) correlation between a ¹⁵N‐tagged nitro group and the adjacent aromatic proton. Once the chemical shift of this proton was determined, we compared the ¹H‐¹³C HSQC spectra of untreated and nitrated samples. All tyrosines were nitrated at ε positions, in agreement to previous analysis by indirect techniques. Notably, the various nitrotyrosine residues show a different dynamic behaviour that is consistent with molecular dynamics computations.     

A Disulfide Bridge Allows for Site‐Selective Binding in Liver Bile Acid Binding Protein Thereby Stabilising the Orientation of Key Amino Acid Side Chains

The presence of a disulfide bridge in liver bile acid binding protein (L‐BABP/S‐S) allows for site‐selective binding of two bile acids, glycochenodeoxycholic (GCDA) and glycocholic acid (GCA), differing only in the presence of a hydroxyl group. The protein form devoid of the disulfide bridge (L‐BABP) binds both bile salts without discriminating ability. We investigate the determinants of the molecular recognition process in the formation of the heterotypic L‐BABP/S‐S complex with GCA and GCDA located in the superficial and inner protein sites, respectively. The comparison of the NMR spectroscopy structure of heterotypic holo L‐BABP/S‐S, the first reported for this protein family, with that of the homotypic L‐BABP complex demonstrates that the introduction of a S–S link between adjacent strands changes the conformation of three key residues, which function as hot‐spot mediators of molecular discrimination. The favoured χ₁ rotameric states (t, g⁺ and g^? for E99, Q100 and E109 residues, respectively) allow the onset of an extended intramolecular hydrogen‐bond network and the consequent stabilisation of the side‐chain orientation of a buried histidine, which is capable of anchoring a specific ligand.