Volume and compressibility differences between protein conformations revealed by high-pressure NMR.

Proteins often interconvert between different conformations in ways critical to their function. Although manipulating such equilibria for biophysical study is often challenging, the application of pressure is a potential route to achieve such control by favoring the population of lower volume states. Here, we use this feature to study the interconversion of ARNT PAS-B Y456T, which undergoes a dramatic +3 slip in the ß-strand register as it switches between two stably folded conformations. Using high-pressure biomolecular NMR approaches, we obtained the first, to our knowledge, quantitative data testing two key hypotheses of this process: the slipped conformation is both smaller and less compressible than the wild-type equivalent, and the interconversion proceeds through a chiefly unfolded intermediate state. Data collected in steady-state pressure and time-resolved pressure-jump modes, including observed pressure-dependent changes in the populations of the two conformers and increased rate of interconversion between conformers, support both hypotheses. Our work exemplifies how these approaches, which can be generally applied to protein conformational switches, can provide unique information that is not easily accessible through other techniques.

Insights into Structural and Dynamical Changes Experienced by Human RNase 6 upon Ligand Binding.

Ribonuclease 6 (RNase 6) is one of eight catalytically active human pancreatic-type RNases that belong to a superfamily of rapidly evolving enzymes. Like some of its human homologues, RNase 6 exhibits host defense properties such as antiviral and antibacterial activities. Recently solved crystal structures of this enzyme in its nucleotide-free form show the conservation of the prototypical kidney-shaped fold preserved among vertebrate RNases, in addition to revealing the presence of a unique secondary active site. In this study, we determine the structural and conformational properties experienced by RNase 6 upon binding to substrate and product analogues. We present the first crystal structures of RNase 6 bound to a nucleotide ligand (adenosine 5'-monophosphate), in addition to RNase 6 bound to phosphate ions. While the enzyme preserves B2 subsite ligand preferences, our results show a lack of typical B2 subsite interactions normally observed in homologous ligand-bound RNases. A comparison of the dynamical properties of RNase 6 in its apo-, substrate-, and product-bound states highlight the unique dynamical properties experienced on time scales ranging from nano- to milliseconds. Overall, our results confirm the specific evolutionary adaptation of RNase 6 relative to its unique catalytic and biological activities.

Ligand Binding Enhances Millisecond Conformational Exchange in Xylanase B2 from Streptomyces lividans.

Xylanases catalyze the hydrolysis of xylan, an abundant carbon and energy source with important commercial ramifications. Despite tremendous efforts devoted to the catalytic improvement of xylanases, success remains limited because of our relatively poor understanding of their molecular properties. Previous reports suggested the potential role of atomic-scale residue dynamics in modulating the catalytic activity of GH11 xylanases; however, dynamics in these studies was probed on time scales orders of magnitude faster than the catalytic time frame. Here, we used nuclear magnetic resonance titration and relaxation dispersion experiments ((15)N-CPMG) in combination with X-ray crystallography and computational simulations to probe conformational motions occurring on the catalytically relevant millisecond time frame in xylanase B2 (XlnB2) and its catalytically impaired mutant E87A from Streptomyces lividans 66. Our results show distinct dynamical properties for the apo and ligand-bound states of the enzymes. The apo form of XlnB2 experiences conformational exchange for residues in the fingers and palm regions of the catalytic cleft, while the catalytically impaired E87A variant displays millisecond dynamics only in the fingers, demonstrating the long-range effect of the mutation on flexibility. Ligand binding induces enhanced conformational exchange of residues interacting with the ligand in the fingers and thumb loop regions, emphasizing the potential role of residue motions in the fingers and thumb loop regions for recognition, positioning, processivity, and/or stabilization of ligands in XlnB2. To the best of our knowledge, this work represents the first experimental characterization of millisecond dynamics in a GH11 xylanase family member. These results offer new insights into the potential role of conformational exchange in GH11 enzymes, providing essential dynamic information to help improve protein engineering and design applications.

Cytosolic galectin-7 impairs p53 functions and induces chemoresistance in breast cancer cells.

BACKGROUND: Resistance to apoptosis induced by anti-cancer drugs is a major obstacle for the treatment of aggressive forms of breast cancer. Galectin-7 (gal-7) was recently shown to be specifically expressed in basal-like but not in luminal subtypes of human breast cancer. METHODS: We generated a mutant form of gal-7 (R74S). Arginine 74 is the structural equivalent of arginine 186 found in human galectin-3. Mutation R186S was previously shown to abolish the biological function of galectin-3. RESULTS: Mutation of arginine 74 induced only limited and local changes to the gal-7 fold. Recombinant forms of R74S and wtgal-7 were also equally effective at forming dimers in solution. Analysis of the thermodynamic parameters by isothermal titration calorimetry (ITC) indicated, however, that binding of lactose to gal-7 was inhibited by the R74S mutation. Using confocal microscopy and electron microscopy, we confirmed the expression of gal-7 in the cytosolic and nuclear compartments of breast cancer cells and the ability of gal-7 to translocate to mitochondria. The mutation at position 74, however, greatly reduced the expression of gal-7 in the nuclear and mitochondrial compartments. Interestingly, cells expressing mutated gal-7 were equally if not even more resistant to drug-induced apoptosis when compared to cells expressing wtgal-7. We also found that both wtgal-7 and R74S inhibited dox-induced PARP-1 cleavage and p53 protein expression. The inhibition of p53 correlated with a decrease in p21 protein expression and CDKN1A mRNA. Furthermore, analysis of nuclear and cytoplasmic fractions showed that both wild type and R74S mutant gal-7 inhibited p53 nuclear translocation, possibly by increasing degradation of cytosolic p53. CONCLUSIONS: These findings pose a challenge to the paradigm that has guided the design of galectin-specific inhibitors for the treatment of cancer. This study suggests that targeting CRD-independent cytosolic gal-7 in breast cancer cells may be a valuable strategy for the treatment of this disease. Our study will thus complement efforts towards improving selectivity of targeted anticancer agents.

Backbone and methyl side-chain resonance assignments of the single chain Fab fragment of trastuzumab.

Trastuzumab is a therapeutic monoclonal antibody developed to target human epidermal growth factor receptor 2 (HER2) present at higher levels in early cancers. Here we report the near complete resonance assignment of trastuzumab-scFab fragment backbone and the methyl groups of isoleucine, leucine and valine residues, as well as their stereo-assignments. The antibody fragment was produced using a single chain approach in Escherichia coli.

Conservation of flexible residue clusters among structural and functional enzyme homologues.

Conformational flexibility between structural ensembles is an essential component of enzyme function. Although the broad dynamical landscape of proteins is known to promote a number of functional events on multiple time scales, it is yet unknown whether structural and functional enzyme homologues rely on the same concerted residue motions to perform their catalytic function. It is hypothesized that networks of contiguous and flexible residue motions occurring on the biologically relevant millisecond time scale evolved to promote and/or preserve optimal enzyme catalysis. In this study, we use a combination of NMR relaxation dispersion, model-free analysis, and ligand titration experiments to successfully capture and compare the role of conformational flexibility between two structural homologues of the pancreatic ribonuclease family: RNase A and eosinophil cationic protein (or RNase 3). In addition to conserving the same catalytic residues and structural fold, both homologues show similar yet functionally distinct clusters of millisecond dynamics, suggesting that conformational flexibility can be conserved among analogous protein folds displaying low sequence identity. Our work shows that the reduced conformational flexibility of eosinophil cationic protein can be dynamically and functionally reproduced in the RNase A scaffold upon creation of a chimeric hybrid between the two proteins. These results support the hypothesis that conformational flexibility is partly required for catalytic function in homologous enzyme folds, further highlighting the importance of dynamic residue sectors in the structural organization of proteins.

Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies.

The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either Komagataella phaffii (Pichia pastoris) or Escherichia coli that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The E. coli approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while K. phaffii secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10-45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-1H-15N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.

Age-dependent B cell autoimmunity to a myelin surface antigen in pediatric multiple sclerosis.

Multiple sclerosis (MS) typically manifests in early to mid adulthood, but there is increasing recognition of pediatric-onset MS, aided by improvements in imaging techniques. The immunological mechanisms of disease are largely unexplored in pediatric-onset MS, in part because studies have historically focused on adult-onset disease. We investigated autoantibodies to myelin surface Ags in a large cohort of pediatric MS cases by flow cytometric labeling of transfectants that expressed different myelin proteins. Although Abs to native myelin oligodendrocyte glycoprotein (MOG) were uncommon among adult-onset patients, a subset of pediatric patients had serum Abs that brightly labeled the MOG transfectant. Abs to two other myelin surface Ags were largely absent. Affinity purification of MOG Abs as well as competition of binding with soluble MOG documented their binding specificity. Such affinity purified Abs labeled myelin and glial cells in human CNS white matter as well as myelinated axons in gray matter. The prevalence of such autoantibodies was highest among patients with a very early onset of MS: 38.7% of patients less than 10 years of age at disease onset had MOG Abs, compared with 14.7% of patients in the 10- to 18-year age group. B cell autoimmunity to this myelin surface Ag is therefore most common in patients with a very early onset of MS.

The Structural Dynamics of Engineered ß-Lactamases Vary Broadly on Three Timescales yet Sustain Native Function.

Understanding the principles of protein dynamics will help guide engineering of protein function: altering protein motions may be a barrier to success or may be an enabling tool for protein engineering. The impact of dynamics on protein function is typically reported over a fraction of the full scope of motional timescales. If motional patterns vary significantly at different timescales, then only by monitoring motions broadly will we understand the impact of protein dynamics on engineering functional proteins. Using an integrative approach combining experimental and in silico methodologies, we elucidate protein dynamics over the entire span of fast to slow timescales (ps to ms) for a laboratory-engineered system composed of five interrelated ß-lactamases: two natural homologs and three laboratory-recombined variants. Fast (ps-ns) and intermediate (ns-µs) dynamics were mostly conserved. However, slow motions (µs-ms) were few and conserved in the natural homologs yet were numerous and widely dispersed in their recombinants. Nonetheless, modified slow dynamics were functionally tolerated. Crystallographic B-factors from high-resolution X-ray structures were partly predictive of the conserved motions but not of the new slow motions captured in our solution studies. Our inspection of protein dynamics over a continuous range of timescales vividly illustrates the complexity of dynamic impacts of protein engineering as well as the functional tolerance of an engineered enzyme system to new slow motions.

Elevated serum inflammatory markers in post-poliomyelitis syndrome.

OBJECTIVES: To determine (i) whether serum inflammatory markers TNFalpha, IL-1beta. IL-6, and leptin are increased in post-poliomyelitis syndrome (PPS) compared to healthy controls; and (ii) whether an association exists between elevated inflammatory markers and clinical parameters in PPS. The cause of PPS is unknown, but abnormal inflammatory responses have been implicated in several small studies. METHODS: Serum inflammatory markers were measured (by Luminex) in 51 PPS patients and 26 normal controls. Clinical parameters assessed included disease duration, muscle strength (Medical Research Council sumscore), fatigue (Fatigue Severity Scale and Multidimensional Fatigue Inventory), and pain (visual analog scale scores). RESULTS: In PPS, TNFalpha levels, as well as IL-6 and leptin were significantly increased compared to controls (Wilcoxon rank-sum test, p=0.03 for TNFalpha, p=0.03 for IL-6, p=0.01 for leptin). The elevated TNFalpha levels in PPS were associated with increased pain due to illness (Spearman correlation coefficient r=0.36, 95% C.I. 0.09 to 0.57) and specifically, with muscle pain (r=0.38, 95% C.I. 0.11 to 0.59). There were no correlations between inflammatory markers in PPS and joint pain, muscle strength, fatigue, or disease duration. CONCLUSIONS: Serum TNFalpha, IL-6 and leptin levels are abnormally increased in PPS patients. Elevated TNFalpha levels appear to be specifically associated with increased muscle pain.

Conservation of Dynamics Associated with Biological Function in an Enzyme Superfamily.

Enzyme superfamily members that share common chemical and/or biological functions also share common features. While the role of structure is well characterized, the link between enzyme function and dynamics is not well understood. We present a systematic characterization of intrinsic dynamics of over 20 members of the pancreatic-type RNase superfamily, which share a common structural fold. This study is motivated by the fact that the range of chemical activity as well as molecular motions of RNase homologs spans over 105 folds. Dynamics was characterized using a combination of nuclear magnetic resonance experiments and computer simulations. Phylogenetic clustering led to the grouping of sequences into functionally distinct subfamilies. Detailed characterization of the diverse RNases showed conserved dynamical traits for enzymes within subfamilies. These results suggest that selective pressure for the conservation of dynamical behavior, among other factors, may be linked to the distinct chemical and biological functions in an enzyme superfamily.

Correction to: Sequence-specific backbone resonance assignments and microsecond timescale molecular dynamics simulation of human eosinophil-derived neurotoxin.

After publication of this article, the authors noticed that a 15N-13C dimension error was unwillingly coded in the 3D NMR spectrum "fid.com" processing script used to perform backbone assignments for this enzyme. The authors noticed that some OBS, CAR and LAB values in the "fid.com" had been switched in the y and z dimensions, probably resulting from a wrong NMRPipe selection when reading the Varian NMR experimental parameters. They have carefully re-processed, re-analyzed, re-assigned, in addition to checking all scripts to evaluate the extent of this processing error on the published assignments. Authors determined that the "fid.com" error resulted in a significant number of incorrect backbone resonance assignments, requiring us to issue corrections in Figs. 2, 3 and 4 of this published manuscript, in addition to Table S1. New versions of these figures and table are provided below. The corresponding BMRB entry has also been revised. The authors note that these modifications do not change the global message, conclusions, and molecular dynamics simulations presented in this article. The authors are grateful to David N. Bernard (INRS) for help with finding and correcting these errors.

Ligand-Induced Variations in Structural and Dynamical Properties Within an Enzyme Superfamily.

Enzyme catalysis is a complex process involving several steps along the reaction coordinates, including substrate recognition and binding, chemical transformation, and product release. Evidence continues to emerge linking the functional and evolutionary role of conformational exchange processes in optimal catalytic activity. Ligand binding changes the conformational landscape of enzymes, inducing long-range conformational rearrangements. Using functionally distinct members of the pancreatic ribonuclease superfamily as a model system, we characterized the structural and conformational changes associated with the binding of two mononucleotide ligands. By combining NMR chemical shift titration experiments with the chemical shift projection analysis (CHESPA) and relaxation dispersion experiments, we show that biologically distinct members of the RNase superfamily display discrete chemical shift perturbations upon ligand binding that are not conserved even in structurally related members. Amino acid networks exhibiting coordinated chemical shift displacements upon binding of the two ligands are unique to each of the RNases analyzed. Our results reveal the contribution of conformational rearrangements to the observed chemical shift perturbations. These observations provide important insights into the contribution of the different ligand binding specificities and effects of conformational exchange on the observed perturbations associated with ligand binding for functionally diverse members of the pancreatic RNase superfamily.

Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily.

In this work, we applied the sequence-based statistical coupling analysis approach to characterize conserved amino acid networks important for biochemical function in the pancreatic-type ribonuclease (ptRNase) superfamily. This superfamily-wide analysis indicates a decomposition of the RNase tertiary structure into spatially distributed yet physically connected networks of co-evolving amino acids, termed sectors. Comparison of this statistics-based description with new NMR experiments data shows that discrete amino acid networks, termed sectors, control the tuning of distinct functional properties in different enzyme homologs. Further, experimental characterization of evolutionarily distant sequences reveals that sequence variation at sector positions can distinguish homologs with a conserved dynamic pattern and optimal catalytic activity from those with altered dynamics and diminished catalytic activities. Taken together, these results provide important insights into the mechanistic design of the ptRNase superfamily, and presents a structural basis for evolutionary tuning of function in functionally diverse enzyme homologs.

Sequence-specific backbone resonance assignments and microsecond timescale molecular dynamics simulation of human eosinophil-derived neurotoxin.

Eight active canonical members of the pancreatic-like ribonuclease A (RNase A) superfamily have been identified in human. All structural homologs share similar RNA-degrading functions, while also cumulating other various biological activities in different tissues. The functional homologs eosinophil-derived neurotoxin (EDN, or RNase 2) and eosinophil cationic protein (ECP, or RNase 3) are known to be expressed and secreted by eosinophils in response to infection, and have thus been postulated to play an important role in host defense and inflammatory response. We recently initiated the biophysical and dynamical investigation of several vertebrate RNase homologs and observed that clustering residue dynamics appear to be linked with the phylogeny and biological specificity of several members. Here we report the 1H, 13C and 15N backbone resonance assignments of human EDN (RNase 2) and its molecular dynamics simulation on the microsecond timescale, providing means to pursue this comparative atomic-scale functional and dynamical analysis by NMR and computation over multiple time frames.

15N, 13C and 1H backbone resonance assignments of an artificially engineered TEM-1/PSE-4 class A ß-lactamase chimera and its deconvoluted mutant.

The widespread use of ß-lactam antibiotics has given rise to a dramatic increase in clinically-relevant ß-lactamases. Understanding the structure/function relation in these variants is essential to better address the ever-growing incidence of antibiotic resistance. We previously reported the backbone resonance assignments of a chimeric protein constituted of segments of the class A ß-lactamases TEM-1 and PSE-4 (Morin et al. in Biomol NMR Assign 4:127-130, 2010. doi: 10.1007/s12104-010-9227-8 ). That chimera, cTEM17m, held 17 amino acid substitutions relative to TEM-1 ß-lactamase, resulting in a well-folded and fully functional protein with increased dynamics. Here we report the (1)H, (13)C and (15)N backbone resonance assignments of chimera cTEM-19m, which includes 19 substitutions and exhibits increased active-site perturbation, as well as one of its deconvoluted variants, as the first step in the analysis of their dynamic behaviours.

Sequence-specific backbone (1)H, (13)C, and (15)N resonance assignments of human ribonuclease 4.

Human ribonuclease 4 (RNase 4) is the most evolutionarily conserved member of the 8 canonical human pancreatic-like RNases, showing more than 90% identity with bovine and porcine homologues. The enzyme displays ribonucleolytic activity with a strong preference for uracil-containing RNA substrates, a feature only shared with human eosinophil derived-neurotoxin (EDN, or RNase 2) and eosinophil cationic protein (ECP, or RNase 3). It is also the shortest member of the human family, with a significantly truncated C-terminal tail. Its unique active-site pocket and high degree of conservation among vertebrates suggest that the enzyme plays a crucial biological function. Here, we report on the (1)H, (13)C and (15)N backbone resonance assignments of RNase 4, providing means to characterize its molecular function at the atomic level by NMR.

Network of long-range concerted chemical shift displacements upon ligand binding to human angiogenin.

Molecular recognition models of both induced fit and conformational selection rely on coupled networks of flexible residues and/or structural rearrangements to promote protein function. While the atomic details of these motional events still remain elusive, members of the pancreatic ribonuclease superfamily were previously shown to depend on subtle conformational heterogeneity for optimal catalytic function. Human angiogenin, a structural homologue of bovine pancreatic RNase A, induces blood vessel formation and relies on a weak yet functionally mandatory ribonucleolytic activity to promote neovascularization. Here, we use the NMR chemical shift projection analysis (CHESPA) to clarify the mechanism of ligand binding in human angiogenin, further providing information on long-range intramolecular residue networks potentially involved in the function of this enzyme. We identify two main clusters of residue networks displaying correlated linear chemical shift trajectories upon binding of substrate fragments to the purine- and pyrimidine-specific subsites of the catalytic cleft. A large correlated residue network clusters in the region corresponding to the V1 domain, a site generally associated with the angiogenic response and structural stability of the enzyme. Another correlated network (residues 40-42) negatively affects the catalytic activity but also increases the angiogenic activity. (15) N-CPMG relaxation dispersion experiments could not reveal the existence of millisecond timescale conformational exchange in this enzyme, a lack of flexibility supported by the very low-binding affinities and catalytic activity of angiogenin. Altogether, the current report potentially highlights the existence of long-range dynamic reorganization of the structure upon distinct subsite binding events in human angiogenin.

Perturbation of the Conformational Dynamics of an Active-Site Loop Alters Enzyme Activity.

The role of internal dynamics in enzyme function is highly debated. Specifically, how small changes in structure far away from the reaction site alter protein dynamics and overall enzyme mechanisms is of wide interest in protein engineering. Using RNase A as a model, we demonstrate that elimination of a single methyl group located >10 Å away from the reaction site significantly alters conformational integrity and binding properties of the enzyme. This A109G mutation does not perturb structure or thermodynamic stability, both in the apo and ligand-bound states. However, significant enhancement in conformational dynamics was observed for the bound variant, as probed over nano- to millisecond timescales, resulting in major ligand repositioning. These results illustrate the large effects caused by small changes in structure on long-range conformational dynamics and ligand specificities within proteins, further supporting the importance of preserving wild-type dynamics in enzyme systems that rely on flexibility for function.

Design of a peptidic inhibitor that targets the dimer interface of a prototypic galectin.

Galectins are small soluble lectins that bind α-galactosides via their carbohydrate recognition domain (CRD). Their ability to dimerize is critical for the crosslinking of glycoprotein receptors and subsequent cellular signaling. This is particularly important in their immunomodulatory role via the induction of T-cell apoptosis. Because galectins play a central role in many pathologies, including cancer, they represent valuable therapeutic targets. At present, most inhibitors have been directed towards the CRD, a challenging task in terms of specificity given the high structural homology of the CRD among galectins. Such inhibitors are not effective at targeting CRD-independent functions of galectins. Here, we report a new class of galectin inhibitors that specifically binds human galectin-7 (hGal-7), disrupts the formation of homodimers, and inhibits the pro-apoptotic activity of hGal-7 on Jurkat T cells. In addition to representing a new means to achieve specificity when targeting galectins, such inhibitors provide a promising alternative to more conventional galectin inhibitors that target the CRD with soluble glycans or other small molecular weight allosteric inhibitors.