Ancestral sequence reconstruction dissects structural and functional differences among eosinophil ribonucleases.

Evolutionarily conserved structural folds can give rise to diverse biological functions, yet predicting atomic-scale interactions that contribute to the emergence of novel activities within such folds remains challenging. Pancreatic-type ribonucleases illustrate this complexity, sharing a core structure that has evolved to accommodate varied functions. In this study, we used ancestral sequence reconstruction to probe evolutionary and molecular determinants that distinguish biological activities within eosinophil members of the RNase 2/3 subfamily. Our investigation unveils functional, structural, and dynamical behaviors that differentiate the evolved ancestral ribonuclease (AncRNase) from its contemporary eosinophil RNase orthologs. Leveraging the potential of ancestral reconstruction for protein engineering, we used AncRNase predictions to design a minimal 4-residue variant that transforms human RNase 2 into a chimeric enzyme endowed with the antimicrobial and cytotoxic activities of RNase 3 members. This work provides unique insights into mutational and evolutionary pathways governing structure, function, and conformational states within the eosinophil RNase subfamily, offering potential for targeted modulation of RNase-associated functions.

Conformational exchange divergence along the evolutionary pathway of eosinophil-associated ribonucleases.

The evolutionary role of conformational exchange in the emergence and preservation of function within structural homologs remains elusive. While protein engineering has revealed the importance of flexibility in function, productive modulation of atomic-scale dynamics has only been achieved on a finite number of distinct folds. Allosteric control of unique members within dynamically diverse structural families requires a better appreciation of exchange phenomena. Here, we examined the functional and structural role of conformational exchange within eosinophil-associated ribonucleases. Biological and catalytic activity of various EARs was performed in parallel to mapping their conformational behavior on multiple timescales using NMR and computational analyses. Despite functional conservation and conformational seclusion to a specific domain, we show that EARs can display similar or distinct motional profiles, implying divergence rather than conservation of flexibility. Comparing progressively more distant enzymes should unravel how this subfamily has evolved new functions and/or altered their behavior at the molecular level.

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.

Nucleotide substrate binding characterization in human pancreatic-type ribonucleases.

Human genome contains a group of more than a dozen similar genes with diverse biological functions including antiviral, antibacterial and angiogenesis activities. The characterized gene products of this group show significant sequence similarity and a common structural fold associated with binding and cleavage of ribonucleic acid (RNA) substrates. Therefore, these proteins have been categorized as members of human pancreatic-type ribonucleases (hRNases). hRNases differ in cell/tissue localization and display distinct substrate binding preferences and a wide range of ribonucleolytic catalytic efficiencies. Limited information is available about structural and dynamical properties that influence this diversity among these homologous RNases. Here, we use computer simulations to characterize substrate interactions, electrostatics and dynamical properties of hRNases 1-7 associated with binding to two nucleotide substrates (ACAC and AUAU). Results indicate that even with complete conservation of active-site catalytic triad associated with ribonucleolytic activity, these enzymes show significant differences in substrate interactions. Detailed characterization suggests that in addition to binding site electrostatic and van der Waals interactions, dynamics of distal regions may also play a role in binding. Another key insight is that a small difference in temperature of 300 K (used in experimental studies) and 310 K (physiological temperature) shows significant changes in enzyme-substrate interactions.

Antibacterial properties of the pituitary adenylate cyclase-activating polypeptide: A new human antimicrobial peptide.

The Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a polycationic, amphiphilic and helical neuropeptide, is well known for its neuroprotective actions and cell penetrating properties. In the present study, we evaluated the potent antibacterial property of PACAP38 and related analogs against various bacterial strains. Interestingly, PACAP38 and related analogs can inhibit the growth of various bacteria including Escherichia coli (JM109), Bacillus subtilis (PY79), and the pathogenic Burkholderia cenocepacia (J2315). Investigation of the mechanism of action suggested that a PACAP metabolite, identified as PACAP(9-38), might indeed be responsible for the observed PACAP38 antibacterial action. Surprisingly, PACAP(9-38), which does not induce haemolysis, exhibits an increased specificity toward Burkholderia cenocepacia J2315 compared to other tested bacteria. Finally, the predisposition of PACAP(9-38) to adopt a π-helix conformation rather than an α-helical conformation like PACAP38 could explain this gain in specificity. Overall, this study has revealed a new function for PACAP38 and related derivatives that can be added to its pleiotropic biological activities. This innovative study could therefore pave the way toward the development of new therapeutic agents against multiresistant bacteria, and more specifically the Burkholderia cenocepacia complex.

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.

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.

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.

Applications of NMR and computational methodologies to study protein dynamics.

Overwhelming evidence now illustrates the defining role of atomic-scale protein flexibility in biological events such as allostery, cell signaling, and enzyme catalysis. Over the years, spin relaxation nuclear magnetic resonance (NMR) has provided significant insights on the structural motions occurring on multiple time frames over the course of a protein life span. The present review article aims to illustrate to the broader community how this technique continues to shape many areas of protein science and engineering, in addition to being an indispensable tool for studying atomic-scale motions and functional characterization. Continuing developments in underlying NMR technology alongside software and hardware developments for complementary computational approaches now enable methodologies to routinely provide spatial directionality and structural representations traditionally harder to achieve solely using NMR spectroscopy. In addition to its well-established role in structural elucidation, we present recent examples that illustrate the combined power of selective isotope labeling, relaxation dispersion experiments, chemical shift analyses, and computational approaches for the characterization of conformational sub-states in proteins and enzymes.

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.

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.

Role of Conformational Motions in Enzyme Function: Selected Methodologies and Case Studies.

It is now common knowledge that enzymes are mobile entities relying on complex atomic-scale dynamics and coordinated conformational events for proper ligand recognition and catalysis. However, the exact role of protein dynamics in enzyme function remains either poorly understood or difficult to interpret. This mini-review intends to reconcile biophysical observations and biological significance by first describing a number of common experimental and computational methodologies employed to characterize atomic-scale residue motions on various timescales in enzymes, and second by illustrating how the knowledge of these motions can be used to describe the functional behavior of enzymes and even act upon it. Two biologically relevant examples will be highlighted, namely the HIV-1 protease and DNA polymerase ß enzyme systems.

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.

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.

Exploring the free energy landscape of a model ß-hairpin peptide and its isoform.

Secondary structural transitions from α-helix to ß-sheet conformations are observed in several misfolding diseases including Alzheimer's and Parkinson's. Determining factors contributing favorably to the formation of each of these secondary structures is therefore essential to better understand these disease states. ß-hairpin peptides form basic components of anti-parallel ß-sheets and are suitable model systems for characterizing the fundamental forces stabilizing ß-sheets in fibrillar structures. In this study, we explore the free energy landscape of the model ß-hairpin peptide GB1 and its E2 isoform that preferentially adopts α-helical conformations at ambient conditions. Umbrella sampling simulations using all-atom models and explicit solvent are performed over a large range of end-to-end distances. Our results show the strong preference of GB1 and the E2 isoform for ß-hairpin and α-helical conformations, respectively, consistent with previous studies. We show that the unfolded states of GB1 are largely populated by misfolded ß-hairpin structures which differ from each other in the position of the ß-turn. We discuss the energetic factors contributing favorably to the formation of α-helix and ß-hairpin conformations in these peptides and highlight the energetic role of hydrogen bonds and non-bonded interactions.

Hydrophobic interactions and hydrogen bonds in ß-sheet formation.

In this study, we investigate interactions of extended conformations of homodimeric peptides made of small (glycine or alanine) and large hydrophobic (valine or leucine) sidechains using all-atom molecular dynamics simulations to decipher driving forces for ß-sheet formation. We make use of a periodic boundary condition setup in which individual peptides are infinitely long and stretched. Dimers adopt ß-sheet conformations at short interpeptide distances (ξ ~ 0.5 nm) and at intermediate distances (~0.8 nm), valine and leucine homodimers assume cross-ß-like conformations with side chains interpenetrating each other. These two states are identified as minima in the potential of mean force. While the number of interpeptide hydrogen bonds increases with decreasing interpeptide distance, the total hydrogen bond number in the system does not change significantly, suggesting that formation of ß-sheet structures from extended conformations is not driven by hydrogen bonds. This is supported by an increase in electrostatic energy at short interpeptide distances. A remarkable correlation between the volume of the system and the total electrostatic energy is observed, further reinforcing the idea that excluding water in proteins comes with an enthalpic penalty. We also discuss microscopic mechanisms accounting for ß-sheet formation based on computed enthalpy and entropy and we show that they are different for peptides with small and large side chains.

Investigation of the Polymeric Properties of α-Synuclein and Comparison with NMR Experiments: A Replica Exchange Molecular Dynamics Study.

Intrinsically disordered proteins (IDPs) have been shown to be involved in a number of cellular functions, in addition to their predominance in diseased states. α-synuclein may be described as one such IDP implicated in the pathology of Parkinson's disease. Understanding the conformational characteristics of the monomeric state of α-synuclein is necessary for understanding the role of the monomer conformation in aggregation. Polymer theories have been applied to investigate the statistical properties of homopolymeric IDPs. Here we use Replica Exchange Molecular Dynamics (REMD) simulations using temperature as a proxy for solvent quality to examine how well these theories developed for homopolymeric chains describe heteropolymeric α-synuclein. Our results indicate that α-synuclein behaves like a homopolymer at the extremes of solvent quality, while in the intermediate solvent regime, the uneven distribution of charged residues along the sequence strongly influences the conformations adopted by the chain. We refine the ensemble extracted from the REMD simulations of α-synuclein, which shows the best qualitative agreement with experiment, by fitting to the experimental NMR Residual Dipolar Couplings (RDCs) and Paramagnetic Relaxation Enhancements (PREs). Our results demonstrate that the detailed shape of the RDC patterns are sensitive to the angular correlations that are local in sequence while longer range anti-correlations which arise from packing constraints affect the RDC magnitudes.

Structural reorganization of alpha-synuclein at low pH observed by NMR and REMD simulations.

alpha-Synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinson's disease. The conversion from monomer to aggregate is complex, and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that alpha-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize alpha-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and replica exchange molecular dynamics simulations indicate that there is a significant structural reorganization within the low-pH ensemble relative to that at neutral pH in terms of long-range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH, there is a very heterogeneous ensemble with transient contacts between the N-terminus and the non-amyloid beta component (NAC); however, at low pH, there is a more homogeneous ensemble that exhibits strong contacts between the NAC and the C-terminus. At both pH values, transient contacts between the N- and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral- and low-pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary-structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral- and low-pH ensembles suggests that the change in long-range interactions between the low- and neutral-pH ensembles, the compaction of the C-terminal region at low pH, and the uneven distribution of charges across the sequence are key to faster aggregation.