Structural and Dynamic-Based Characterization of the Recognition Patterns of E7 and TRP-2 Epitopes by MHC Class I Receptors through Computational Approaches.

A detailed comprehension of MHC-epitope recognition is essential for the design and development of new antigens that could be effectively used in immunotherapy. Yet, the high variability of the peptide together with the large abundance of MHC variants binding makes the process highly specific and large-scale characterizations extremely challenging by standard experimental techniques. Taking advantage of the striking predictive accuracy of AlphaFold, we report a structural and dynamic-based strategy to gain insights into the molecular basis that drives the recognition and interaction of MHC class I in the immune response triggered by pathogens and/or tumor-derived peptides. Here, we investigated at the atomic level the recognition of E7 and TRP-2 epitopes to their known receptors, thus offering a structural explanation for the different binding preferences of the studied receptors for specific residues in certain positions of the antigen sequences. Moreover, our analysis provides clues on the determinants that dictate the affinity of the same epitope with different receptors. Collectively, the data here presented indicate the reliability of the approach that can be straightforwardly extended to a large number of related systems.

Atomic-Level View of the Functional Transition in Vertebrate Hemoglobins: The Case of Antarctic Fish Hbs.

Tetrameric hemoglobins (Hbs) are prototypal systems for studies aimed at unveiling basic structure-function relationships as well as investigating the molecular/structural basis of adaptation of living organisms to extreme conditions. However, a chronological analysis of decade-long studies conducted on Hbs is illuminating on the difficulties associated with the attempts of gaining functional insights from static structures. Here, we applied molecular dynamics (MD) simulations to explore the functional transition from the T to the R state of the hemoglobin of the Antarctic fish Trematomus bernacchii (HbTb). Our study clearly demonstrates the ability of the MD technique to accurately describe the transition of HbTb from the T to R-like states, as shown by a number of global and local structural indicators. A comparative analysis of the structural states that HbTb assumes in the simulations with those detected in previous MD analyses conducted on HbA (human Hb) highlights interesting analogies (similarity of the transition pathway) and differences (distinct population of intermediate states). In particular, the ability of HbTb to significantly populate intermediate states along the functional pathway explains the observed propensity of this protein to assume these structures in the crystalline state. It also explains some functional data reported on the protein that indicate the occurrence of other functional states in addition to the canonical R and T ones. These findings are in line with the emerging idea that the classical two-state view underlying tetrameric Hb functionality is probably an oversimplification and that other structural states play important roles in these proteins. The ability of MD simulations to accurately describe the functional pathway in tetrameric Hbs suggests that this approach may be effectively applied to unravel the molecular and structural basis of Hbs exhibiting peculiar functional properties as a consequence of the environmental adaptation of the host organism.

Copper (I) or (II) Replacement of the Structural Zinc Ion in the Prokaryotic Zinc Finger Ros Does Not Result in a Functional Domain.

A strict interplay is known to involve copper and zinc in many cellular processes. For this reason, the results of copper's interaction with zinc binding proteins are of great interest. For instance, copper interferences with the DNA-binding activity of zinc finger proteins are associated with the development of a variety of diseases. The biological impact of copper depends on the chemical properties of its two common oxidation states (Cu(I) and Cu(II)). In this framework, following the attention addressed to unveil the effect of metal ion replacement in zinc fingers and in zinc-containing proteins, we explore the effects of the Zn(II) to Cu(I) or Cu(II) replacement in the prokaryotic zinc finger domain. The prokaryotic zinc finger protein Ros, involved in the horizontal transfer of genes from A. tumefaciens to a host plant infected by it, belongs to a family of proteins, namely Ros/MucR, whose members have been recognized in different bacteria symbionts and pathogens of mammals and plants. Interestingly, the amino acids of the coordination sphere are poorly conserved in most of these proteins, although their sequence identity can be very high. In fact, some members of this family of proteins do not bind zinc or any other metal, but assume a 3D structure similar to that of Ros with the residues replacing the zinc ligands, forming a network of hydrogen bonds and hydrophobic interactions that surrogates the Zn-coordinating role. These peculiar features of the Ros ZF domain prompted us to study the metal ion replacement with ions that have different electronic configuration and ionic radius. The protein was intensely studied as a perfectly suited model of a metal-binding protein to study the effects of the metal ion replacement; it appeared to tolerate the Zn to Cd substitution, but not the replacement of the wildtype metal by Ni(II), Pb(II) and Hg(II). The structural characterization reported here gives a high-resolution description of the interaction of copper with Ros, demonstrating that copper, in both oxidation states, binds the protein, but the replacement does not give rise to a functional domain.

High-Resolution Conformational Analysis of RGDechi-Derived Peptides Based on a Combination of NMR Spectroscopy and MD Simulations.

The crucial role of integrin in pathological processes such as tumor progression and metastasis formation has inspired intense efforts to design novel pharmaceutical agents modulating integrin functions in order to provide new tools for potential therapies. In the past decade, we have investigated the biological proprieties of the chimeric peptide RGDechi, containing a cyclic RGD motif linked to an echistatin C-terminal fragment, able to specifically recognize αvß3 without cross reacting with αvß5 and αIIbß3 integrin. Additionally, we have demonstrated using two RGDechi-derived peptides, called RGDechi1-14 and ψRGDechi, that chemical modifications introduced in the C-terminal part of the peptide alter or abolish the binding to the αvß3 integrin. Here, to shed light on the structural and dynamical determinants involved in the integrin recognition mechanism, we investigate the effects of the chemical modifications by exploring the conformational space sampled by RGDechi1-14 and ψRGDechi using an integrated natural-abundance NMR/MD approach. Our data demonstrate that the flexibility of the RGD-containing cycle is driven by the echistatin C-terminal region of the RGDechi peptide through a coupling mechanism between the N- and C-terminal regions.

Structural Model for Recruitment of RIT1 to the LZTR1 E3 Ligase: Evidences from an Integrated Computational Approach.

Leucine-zipper transcription regulator 1 (LZTR1) is a highly mutated tumor suppressor gene, involved in the pathogenesis of several cancer types and developmental disorders. In proteasomal degradation, it acts as an adaptor protein responsible for the recognition and recruitment of substrates to be ubiquitinated in Cullin3-RING ligase E3 (CRL3) machinery. LZTR1 belongs to the BTB-Kelch family, a multi-domain protein where the Kelch propeller plays as the substrate recognition region and for which no experimental structure has been solved. Recently, large effort mutational analyses pointed to the role of disease-associated LZTR1 mutations in the RAS/MAPK signaling pathway and RIT1, a small Ras-related GTPase protein, has been identified by mass spectroscopy to interact with LZTR1. Hence, a better understanding of native structure, molecular mechanism, and substrate specificity would help clarifying the role of LZTR1 in pathological diseases, thus promoting advancement in the development of novel therapeutic strategies. Here, we address the interaction model between adaptor LZTR1 and substrate RIT1 by applying an integrated computational approach, including molecular modeling and docking techniques. We observe that the interaction model LZTR1-RIT1 is stabilized by an electrostatic bond network established between the two protein surfaces, which is reminiscent of homologous ubiquitin ligases complexes. Then, running MD simulations, we characterize differential conformational dynamics of the multi-domain LZTR1, offering interesting implications on the mechanistic role of specific point mutations. We identify G248R and R283Q as damaging mutations involved in the recognition process of the substrate RIT1 and R412C as a possible allosteric mutation from the Kelch to the C-term BTB-domain. Our findings provide important structural insights on targeting CRL3s for drug discovery.

Chemical Perturbation of Oncogenic Protein Folding: from the Prediction of Locally Unstable Structures to the Design of Disruptors of Hsp90-Client Interactions.

Protein folding quality control in cells requires the activity of a class of proteins known as molecular chaperones. Heat shock protein-90 (Hsp90), a multidomain ATP driven molecular machine, is a prime representative of this family of proteins. Interactions between Hsp90, its co-chaperones, and client proteins have been shown to be important in facilitating the correct folding and activation of clients. Hsp90 levels and functions are elevated in tumor cells. Here, we computationally predict the regions on the native structures of clients c-Abl, c-Src, Cdk4, B-Raf and Glucocorticoid Receptor, that have the highest probability of undergoing local unfolding, despite being ordered in their native structures. Such regions represent potential ideal interaction points with the Hsp90-system. We synthesize mimics spanning these regions and confirm their interaction with partners of the Hsp90 complex (Hsp90, Cdc37 and Aha1) by Nuclear Magnetic Resonance (NMR). Designed mimics selectively disrupt the association of their respective clients with the Hsp90 machinery, leaving unrelated clients unperturbed and causing apoptosis in cancer cells. Overall, selective targeting of Hsp90 protein-protein interactions is achieved without causing indiscriminate degradation of all clients, setting the stage for the development of therapeutics based on specific chaperone:client perturbation.

Mechanistic Model for the Hsp90-Driven Opening of Human Argonaute.

The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA-mediated gene silencing. Loading of small RNAs into Argonaute (Ago), the key player protein in the process, has been shown to depend on the Hsp90 chaperone machinery. Experimental single-molecule data indicate that ATP binding to the chaperone facilitates the conformational changes leading to the open state of Ago essential to form a complex with small-RNA duplexes. Yet, no atomic-level description of the dynamic mechanisms and protein-protein interactions underpinning Hsp90-mediated Ago conformational activation is available. Here we investigate the functionally oriented structural and dynamic features of Hsp90-human Ago (hAgo2) complexes in different ligand states by integrating protein-protein docking techniques, all-atom MD simulations, and novel methods of analysis of protein internal dynamics and energetics. On this basis, we develop a structural-dynamic model of the mechanisms underlying the chaperone-assisted human RISC assembly. Our approach unveils the large conformational variability displayed by hAgo2 in the unbound vs the Hsp90-bound states. In this context, several hAgo2 states are found to coexist in isolation, while Hsp90 selects and stabilizes the active form. Hsp90 binding modulates the conformational plasticity of hAgo2 (favoring its opening) by modifying the patterns of hAgo2 intramolecular interactions. Finally, we identify a series of experimentally verifiable key sites that can be mutated to modulate Hsp90-mediated hAgo2 conformational response and ability to bind RNA.

Cooperative Binding of the Cationic Porphyrin Tris-T4 Enhances Catalytic Activity of 20S Proteasome Unveiling a Complex Distribution of Functional States.

The present study provides new evidence that cationic porphyrins may be considered as tunable platforms to interfere with the structural "key code" present on the 20S proteasome α-rings and, by consequence, with its catalytic activity. Here, we describe the functional and conformational effects on the 20S proteasome induced by the cooperative binding of the tri-cationic 5-(phenyl)-10,15,20-(tri N-methyl-4-pyridyl) porphyrin (Tris-T4). Our integrated kinetic, NMR, and in silico analysis allowed us to disclose a complex effect on the 20S catalytic activity depending on substrate/porphyrin concentration. The analysis of the kinetic data shows that Tris-T4 shifts the relative populations of the multiple interconverting 20S proteasome conformations leading to an increase in substrate hydrolysis by an allosteric pathway. Based on our Tris-T4/h20S interaction model, Tris-T4 is able to affect gating dynamics and substrate hydrolysis by binding to an array of negatively charged and hydrophobic residues present on the protein surface involved in the 20S molecular activation by the regulatory proteins (RPs). Accordingly, despite the fact that Tris-T4 also binds to the α3ΔN mutant, allosteric modulation is not observed since the molecular mechanism connecting gate dynamics with substrate hydrolysis is impaired. We envisage that the dynamic view of the 20S conformational equilibria, activated through cooperative Tris-T4 binding, may work as a simplified model for a better understanding of the intricate network of 20S conformational/functional states that may be mobilized by exogenous ligands, paving the way for the development of a new generation of proteasome allosteric modulators.

The Importance of Detail: How Differences in Ligand Structures Determine Distinct Functional Responses in Integrin αv ß3.

Ligand-based control of protein functional motions can provide novel opportunities in the study of fundamental biological mechanisms and in the development of novel therapeutics. In this work we addressed the ligand-based modulation of integrin functions. Inhibitors of integrin αv ß3 are interesting anticancer agents but their molecular mechanisms are still unclear: Peptides and peptidomimetics characterized by the Arg-Gly-Asp (RGD) or isoAsp-Gly-Arg (isoDGR) binding motifs have shown controversial agonist/antagonist effects. We have investigated the differential mechanisms of integrin activation/deactivation by three distinct ligands (cyclo-RGDf(NMe)V (Cilengitide), cyclo[DKP3-RGD], cyclo[DKP3-isoDGR]; DKP=diketopiperazine) through a comparative analysis of ligand-controlled protein internal dynamics: Although RGD facilitates the onset of dynamic states leading to activation, isoDGR induces a diffuse rigidification of the complex consistent with antagonist activities. Computational predictions have been experimentally probed by showing that the antibody AP5, which is capable of recognizing the active form of integrin, binds specifically to the RGD complexes and not to the isoDGR complex, which supports opposite functional roles of the two motifs targeting the same binding site.

Exploring the antimalarial potential of the methoxy-thiazinoquinone scaffold: Identification of a new lead candidate.

A small library of antiplasmodial methoxy-thiazinoquinones, rationally designed on the model of the previously identified hit 1, has been prepared by a simple and inexpensive procedure. The synthetic derivatives have been subjected to in vitro pharmacological screening, including antiplasmodial and toxicity assays. These studies afforded a new lead candidate, compound 9, endowed with higher antiplasmodial potency compared to 1, a good selectivity index when tested against a panel of mammalian cells, no toxicity against RBCs, a synergistic antiplasmodial action in combination with dihydroartemisinin, and a promising inhibitory activity on stage V gametocyte growth. Computational studies provided useful insights into the structural requirements needed for the antiplasmodial activity of thiazinoquinone compounds and on their putative mechanism of action.

High Affinity vs. Native Fibronectin in the Modulation of αvß3 Integrin Conformational Dynamics: Insights from Computational Analyses and Implications for Molecular Design.

Understanding how binding events modulate functional motions of multidomain proteins is a major issue in chemical biology. We address several aspects of this problem by analyzing the differential dynamics of αvß3 integrin bound to wild type (wtFN10, agonist) or high affinity (hFN10, antagonist) mutants of fibronectin. We compare the dynamics of complexes from large-scale domain motions to inter-residue coordinated fluctuations to characterize the distinctive traits of conformational evolution and shed light on the determinants of differential αvß3 activation induced by different FN sequences. We propose an allosteric model for ligand-based integrin modulation: the conserved integrin binding pocket anchors the ligand, while different residues on the two FN10's act as the drivers that reorganize relevant interaction networks, guiding the shift towards inactive (hFN10-bound) or active states (wtFN10-bound). We discuss the implications of results for the design of integrin inhibitors.

Structural Characterization of the Lactobacillus Plantarum FlmC Protein Involved in Biofilm Formation.

Lactobacillus plantarum is one of the most predominant species in the human gut microbiota of healthy individuals. We have previously characterized some probiotic features of L. plantarum LM3, as the high resistance to different stress, the binding ability toward some extracellular matrix proteins and plasminogen and the immunomodulatory role of the surface expressed adhesin EnoA1. We have also identified the flmA, flmB and flmC genes, coding for putative proteins named FlmA, FlmB and FlmC, whose null mutations partially impaired biofilm development; the L. plantarum LM3⁻6 strain, carrying a deletion in flmC, showed a high rate of autolysis, supporting the hypothesis that FlmC might be involved in cell wall integrity. Here, we report the in-silico characterization of ΔTM-FlmC, a portion of the FlmC protein. The protein has been also expressed, purified and characterized by means of CD spectroscopy, ICP-mass and UHPLC-HRMS. The obtained experimental data validated the predicted model unveiling also the presence of a bound lipid molecule and of a Mg(II) ion. Overall, we provide strong evidences that ΔTM-FlmC belongs to the LytR-CpsA-Psr (LCP) family of domains and is involved in cell envelope biogenesis.

Structural Stability and Flexibility Direct the Selection of Activating Mutations in Epidermal Growth Factor Receptor Kinase.

Herein we investigate the potential of novel methods of molecular dynamics analysis to provide information on the key factors that underlie the preferential localization and the effects of mutations modulating protein activities. Epidermal growth factor receptor (EGFR) kinases are selected as a test case. The combined analysis of protein energetics and internal dynamics indicates a clear polarization in the native protein, whereby a highly stable and ordered scaffold in one domain, namely the C-lobe, is combined to a flexible and loosely stabilized domain, the N-lobe. The subdivision in two portions with different properties directs the presence of point mutations mainly to the N-lobe. This allows modulating protein flexibility so that the protein can more efficiently sample the conformations necessary for substrate recognition, while leaving the stability of the protein unperturbed. In this context, comparative simulations of EGFR in the wild type sequence and in the presence of the activating oncogenic mutation G719S reveal flexibility changes in several key regions, involving in particular the part of the kinase devoted to the regulation of substrate recognition (regulatory core) and an increase in the number of stabilizing interactions in the N-lobe for the activated mutant. Our approaches represent a promising and simple strategy toward rationalizing the effects of mutations in modulating enzymatic activities.

Molecular basis of substrate-induced permeation by an amino acid antiporter.

Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the "5 + 5 inverted repeat" fold found in other families like the Na(+)-coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg(+)-bound occluded. However, the role of Arg(+) during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg(+)-bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg(+)-guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.

The Action of Chemical Denaturants: From Globular to Intrinsically Disordered Proteins.

Proteins perform their many functions by adopting either a minimal number of strictly similar conformations, the native state, or a vast ensemble of highly flexible conformations. In both cases, their structural features are highly influenced by the chemical environment. Even though a plethora of experimental studies have demonstrated the impact of chemical denaturants on protein structure, the molecular mechanism underlying their action is still debated. In the present review, after a brief recapitulation of the main experimental data on protein denaturants, we survey both classical and more recent interpretations of the molecular basis of their action. In particular, we highlight the differences and similarities of the impact that denaturants have on different structural classes of proteins, i.e., globular, intrinsically disordered (IDP), and amyloid-like assemblies. Particular attention has been given to the IDPs, as recent studies are unraveling their fundamental importance in many physiological processes. The role that computation techniques are expected to play in the near future is illustrated.

A Protein Data Bank survey of multimodal binding of thiocyanate to proteins: Evidence for thiocyanate promiscuity.

Over the last one and half century, a myriad of studies has demonstrated that Hofmeister ions have a major impact on protein stability and solubility. Nevertheless, the definition of the physico-chemical basis of their activity has proved to be highly challenging and controversial. Here, by exploiting the enormous information content of the Protein Data Bank, we explored the binding to proteins of thiocyanate, the anion of the series exerting the highest solubilization/destabilization effects. The survey, which led to the identification and characterization of 712 thiocyanate binding sites, provides a comprehensive and atomic-level view of the varied interactions that the ion forms with proteins. The inspection of these sites highlights a limited tendency of thiocyanate to interact with structured water molecules, in line with the reported poor hydration of the ion. On the other hand, the thiocyanate makes interactions with protein nonpolar moieties, especially with the backbone Cα atom. In as many as 104 cases, the ion exclusively makes nonpolar contacts. In conclusion, these findings suggest that the ability of thiocyanate to bind all types of protein exposed patches may lead to the formation of a negatively charged electrostatic barrier that could prevent protein-protein aggregation and promote protein solubility. Moreover, the denaturing action of thiocyanate may be ascribed to its ability to establish multiple attractive interactions with protein surfaces.

The dynamics of t1 adenosine binding on human Argonaute 2: Understanding recognition with conformational selection.

The control of expression in genetic regulation is a fundamental process for cell life. In RNA-mediated silencing, human Argonaute-2 protein (hAgo2) uses sequence information encoded in small RNAs (guide) to identify complementary sites in messenger RNAs (target) for repression. The specificity of this molecular recognition lies at the basis of the mechanisms that control the expression of thousands of genes, which necessarily requires a fine tuning of complex events. Among these, the binding of the first nucleotide of the target RNA (t1) is emerging as an important modulator of hAgo2-mediated machinery. Using atomistic molecular dynamics-derived analyses, we address the mechanism behind t1-dependent regulation and study the impact of different t1 nucleotides (t1A, t1C, t1G, t1U) on the conformational dynamics of both hAgo2 and guide-target RNAs. Only when an adenine is found at this position, t1 directly interacts with a specific hAgo2 binding pocket, favoring the stabilization of target binding. Our findings show that hAgo2 exploits a dynamic recognition mechanism of the t1-target thanks to a modulation of RNA conformations. Here, t1-adenine is the only nucleobase endowed with a dual binding mode: a T-shape and a co-planar conformation, respectively, orthogonal and parallel to the following base-pairs of guide-target duplex. This triggers a composite set of molecular interactions that stabilizes distinctive conformational ensembles. Our comparative analyses show characteristic traits of local and global dynamic interplay between hAgo2 and the RNA molecules and highlight how t1A binding acts as a molecular switch for target recognition and complex stabilization. Implications for future mechanistic studies are discussed.

A Structure-Based Mechanism for the Denaturing Action of Urea, Guanidinium Ion and Thiocyanate Ion.

An exhaustive analysis of all the protein structures deposited in the Protein Data Bank, here performed, has allowed the identification of hundredths of protein-bound urea molecules and the structural characterization of such binding sites. It emerged that, even though urea molecules are largely involved in hydrogen bonds with both backbone and side chains, they are also able to make van der Waals contacts with nonpolar moieties. As similar findings have also been previously reported for guanidinium and thiocyanate, this observation suggests that promiscuity is a general property of protein denaturants. Present data provide strong support for a mechanism based on the protein-denaturant direct interactions with a denaturant binding model to equal and independent sites. In this general framework, our investigations also highlight some interesting insights into the different denaturing power of urea compared to guanidinium/thiocyanate.

A novel approach for studying receptor-ligand interactions on living cells surface by using NUS/T1ρ-NMR methodologies combined with computational techniques: The RGDechi15D-αvß5 integrin complex.

Structural investigations of receptor-ligand interactions on living cells surface by high-resolution Nuclear Magnetic Resonance (NMR) are problematic due to their short lifetime, which often prevents the acquisition of experiments longer than few hours. To overcome these limitations, we developed an on-cell NMR-based approach for exploring the molecular determinants driving the receptor-ligand recognition mechanism under native conditions. Our method relies on the combination of high-resolution structural and dynamics NMR data with Molecular Dynamics simulations and Molecular Docking studies. The key point of our strategy is the use of Non Uniform Sampling (NUS) and T1ρ-NMR techniques to collect atomic-resolution structural and dynamics information on the receptor-ligand interactions with living cells, that can be used as conformational constraints in computational studies. In fact, the application of these two NMR methodologies allows to record spectra with high S/N ratio and resolution within the lifetime of cells. In particular, 2D NUS [1H-1H] trNOESY spectra are used to explore the ligand conformational changes induced by receptor binding; whereas T1ρ-based experiments are applied to characterize the ligand binding epitope by defining two parameters: T1ρ Attenuation factor and T1ρ Binding Effect. This approach has been tested to characterize the molecular determinants regulating the recognition mechanism of αvß5-integrin by a selective cyclic binder peptide named RGDechi15D. Our data demonstrate that the developed strategy represents an alternative in-cell NMR tool for studying, at atomic resolution, receptor-ligand recognition mechanism on living cells surface. Additionally, our application may be extremely useful for screening of the interaction profiling of drugs with their therapeutic targets in their native cellular environment.

Role of the conformational versatility of the neurotrophin N-terminal regions in their recognition by Trk receptors.

Neurotrophins (NTs) represent a family of proteins that play an important role in the survival, development, and function of neurons. Extensive efforts are currently being made to develop small molecules endowed with agonist or antagonist NT activity. The structurally versatile N-termini of these proteins are considered regions of interest for the design of new molecules. By combining experimental and computational approaches, we analyzed the intrinsic conformational preferences of the N-termini of two of the most important NTs: NGF (NGF-Nter) and NT4 (NT4-Nter). Circular dichroism spectra clearly indicate that both peptides show a preference for random coil states. Because this finding does not preclude the possibility that structured forms may occur in solution as minor conformational states, we performed molecular-dynamics simulations to gain insights into the structural features of populated species. In line with the circular dichroism analysis, the simulations show a preference for unstructured states for both peptides. However, the simulations also show that for NT4-Nter, and to a lesser extent for NGF-Nter, helical conformations, which are required for binding to the Trk receptor, are present in the repertoire of structures that are intrinsically accessible to these peptides. Accordingly, molecular recognition of NTs by the Trk receptor is accomplished by the general mechanism known as population shift. These findings provide a structural rationale for the observed activity of synthetic peptides based on these NT regions. They also suggest strategies for the development of biologically active peptide-based compounds.