Helical Propensity Research Articles

Protein disorder-to-order transition enhances the nucleosome-binding affinity of H1.

Intrinsically disordered proteins are crucial elements of chromatin heterogenous organization. While disorder in the histone tails enables a large variation of inter-nucleosome arrangements, disorder within the chromatin-binding proteins facilitates promiscuous binding to a wide range of different molecular targets, consistent with structural heterogeneity. Among the partially disordered chromatin-binding proteins, the H1 linker histone influences a myriad of chromatin characteristics including compaction, nucleosome spacing, transcription regulation, and the recruitment of other chromatin regulating proteins. Although it is now established that the long C-terminal domain (CTD) of H1 remains disordered upon nucleosome binding and that such disorder favours chromatin fluidity, the structural behaviour and thereby the role/function of the N-terminal domain (NTD) within chromatin is yet unresolved. On the basis of microsecond-long parallel-tempering metadynamics and temperature-replica exchange atomistic molecular dynamics simulations of different H1 NTD subtypes, we demonstrate that the NTD is completely unstructured in solution but undergoes an important disorder-to-order transition upon nucleosome binding: it forms a helix that enhances its DNA binding ability. Further, we show that the helical propensity of the H1 NTD is subtype-dependent and correlates with the experimentally observed binding affinity of H1 subtypes, suggesting an important functional implication of this disorder-to-order transition.

Interaction between the scaffold proteins CBP by IQGAP1 provides an interface between gene expression and cytoskeletal activity

Crosstalk between cellular pathways is often mediated through scaffold proteins that function as platforms for the assembly of signaling complexes. Based on yeast two-hybrid analysis, we report here the interaction between two complex scaffold proteins, CREB-binding protein (CBP) and the Ras GTPase-activating-like protein 1 (IQGAP1). Dissection of the interaction between the two proteins reveals that the central, thus far uncharacterized, region of IQGAP1 interacts with the HAT domain and the C-terminal intrinsically disordered region of CBP (termed ID5). Structural analysis of ID5 by solution NMR spectroscopy and SAXS reveals the presence of two regions with pronounced helical propensity. The ID5 region(s) involved in the interaction of nanomolar affinity were delineated by solution NMR titrations and pull-down assays. Moreover, we found that IQGAP1 acts as an inhibitor of the histone acetyltransferase (HAT) activity of CBP. In in vitro assays, the CBP-binding region of IQGAP1 positively and negatively regulates the function of HAT proteins of different families including CBP, KAT5 and PCAF. As many signaling pathways converge on CBP and IQGAP1, their interaction provides an interface between transcription regulation and the coordination of cytoskeleton. Disruption or alteration of the interaction between these scaffold proteins may lead to cancer development or metastatic processes, highlighting the importance of this interaction.

Stapling a G-quadruplex specific peptide

G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure and the RHAU helicase has been identified to have high specificity for recognition of parallel-stranded G4s. We have designed and synthesized two stapled peptide analogues of the G4-specfic motif of RHAU, which preserve the G4 binding ability. Characterization of these peptides identified the stapled variants to exhibit higher helical formation propensity in aqueous buffer in comparison to the native RHAU sequence. Moreover, the stapled peptides exhibit superior enzymatic stability towards α-chymotrypsin. Our stapled RHAU peptides can serve as a new tool for targeting G4 nucleic acid structures.

Usefulness of medium-angle X-ray scattering for structural characterization of flexible proteins studied by computer simulations

It is widely accepted that disordered regions in proteins, part of which takes predominantly α-helical conformation to a varying degree, play a critical role in biological function. Structural analysis of these flexible proteins is, however, not straightforward because existing methods to characterize the structural features of the disordered regions are not applicable to all the proteins with various sizes and physicochemical properties. In this study, to gain information on the helical propensity in the disordered regions as well as the overall three-dimensional structures of the proteins, usefulness of the combined small- and medium-angle X-ray scattering (SMAXS), which provides structural information at higher spatial resolution than commonly-used small-angle X-ray scattering, was investigated using computer simulations. For this purpose, various conformations of a protein that consists of two well-folded domains connected by a flexible linker were generated while the linker was modeled either to contain several α-helices or as random coils. The differences in the SMAXS scattering curves of these models were then evaluated. Analysis showed that the SMAXS curves allow to distinguish α-helices from random coils if at least ∼20% of all the residues in the proteins contributes to the helical formation in the disordered region, suggesting that structural analysis using SMAXS will be useful not only to obtain the three-dimensional domain organizations but also to gain information on the helical propensity in the disordered regions. This would lead to more accurate interpretation of reaction kinetics that the flexible proteins are involved in.

Unraveling Allostery in a Knotted Minimal Methyltransferase by NMR Spectroscopy

The methyltransferases that belong to the SpoU-TrmD family contain trefoil knots in their backbone fold. Recent structural dynamic and binding analyses of both free and bound homologs indicate that the knot within the polypeptide backbone plays a significant role in the biological activity of the molecule. The knot loops form the S-adenosyl-methionine (SAM)-binding pocket as well as participate in SAM binding and catalysis. Knots contain both at once a stable core as well as moving parts that modulate long-range motions. Here, we sought to understand allosteric effects modulated by the knotted topology. Uncovering the residues that contribute to these changes and the functional aspects of these protein motions are essential to understanding the interplay between the knot, activation of the methyltransferase, and the implications in RNA interactions. The question we sought to address is as follows: How does the knot, which constricts the backbone as well as forms the SAM-binding pocket with its three distinctive loops, affect the binding mechanism? Using a minimally tied trefoil protein as the framework for understanding the structure–function roles, we offer an unprecedented view of the conformational mechanics of the knot and its relationship to the activation of the ligand molecule. Focusing on the biophysical characterization of the knot region by NMR spectroscopy, we identify the SAM-binding region and observe changes in the dynamics of the loops that form the knot. Importantly, we also observe long-range allosteric changes in flanking helices consistent with winding/unwinding in helical propensity as the knot tightens to secure the SAM cofactor.

Odd-even alternations in helical propensity of a homologous series of hydrocarbons.

Odd and even homologues of some n-alkane-based systems are known to exhibit notably different trends in solid-state properties; a well-known illustration is the zigzag plot of their melting point versus chain length. Odd-even effects in the solid state often arise from intermolecular interactions that involve fully extended molecules. These effects have also been observed in less condensed phases, such as self-assembled monolayers; however, the origins of these effects in such systems can be difficult to determine. Here we combined NMR and computational analysis to show that all-syn contiguously methyl-substituted hydrocarbons, with chain lengths from C6 to C11, exhibit a dramatic odd-even effect in helical propensity. The even- and odd-numbered hydrocarbons populate regular and less-controlled helical conformations, respectively. This knowledge will guide the design of helical hydrocarbons as rigid scaffolds or as hydrophobic components in soft materials.

Choice of Force Field for Proteins Containing Structured and Intrinsically Disordered Regions

Biomolecular force fields optimized for globular proteins fail to properly reproduce properties of intrinsically disordered proteins. In particular, parameters of the water model need to be modified to improve applicability of the force fields to both ordered and disordered proteins. Here, we compared performance of force fields recommended for intrinsically disordered proteins in molecular dynamics simulations of three proteins differing in the content of ordered and disordered regions (two proteins consisting of a well-structured domain and of a disordered region with and without a transient helical motif and one disordered protein containing a region of increased helical propensity). The obtained molecular dynamics trajectories were used to predict measurable parameters, including radii of gyration of the proteins and chemical shifts, residual dipolar couplings, paramagnetic relaxation enhancement, and NMR relaxation data of their individual residues. The predicted quantities were compared with experimental data obtained within this study or published previously. The results showed that the NMR relaxation parameters, rarely used for benchmarking, are particularly sensitive to the choice of force-field parameters, especially those defining the water model. Interestingly, the TIP3P water model, leading to an artificial structural collapse, also resulted in unrealistic relaxation properties. The TIP4P-D water model, combined with three biomolecular force-field parameters for the protein part, significantly improved reliability of the simulations. Additional analysis revealed only one particular force field capable of retaining the transient helical motif observed in NMR experiments. The benchmarking protocol used in our study, being more sensitive to imperfections than the commonly used tests, is well suited to evaluate the performance of newly developed force fields.

Backbone Cyclization and Dimerization of LL-37-Derived Peptides Enhance Antimicrobial Activity and Proteolytic Stability.

Can antimicrobial activity and peptide stability of alpha-helical peptides be increased by making them into dimers and macrocycles? Here, we explore that concept by using KR-12 as the starting point for peptide engineering. KR-12 has previously been determined as the minimalized antimicrobial fragment of the human host defense peptide LL-37. Backbone-cyclized KR-12 dimers, tethered by linkers of two to four amino acid residues, were synthesized and their antimicrobial activity, proteolytic stability and structures characterized. A modified KR-12 sequence, with substitutions at previously identified key residues, were also included in the screening panel. The backbone cyclized KR-12 dimers showed improved antimicrobial activity and increased stability compared to monomeric KR-12. The most active cyclic dimer displayed 16-fold higher antibacterial activity compared to KR-12 against Pseudomonas aeruginosa and Staphylococcus aureus, and 8-fold increased fungicidal activity against Candida albicans. It also showed increased hemolytic and cytotoxic activity. Enhanced antimicrobial activity coincided with increased membrane permeabilization of liposomes with one distinct discrepancy: monomeric KR-12 was much less disruptive of liposomes with bacterial lipid composition compared to liposomes from fungal lipid extract. Circular dichroism showed that the four-residue linked most active cyclic dimer had 65% helical content when bound to lyso-phosphatidylglycerol micelles, indicating that the helical propensity of the parent peptide is maintained in the new macrocyclic form. In conclusion, the current work on KR-12 suggests that dimerization together with backbone cyclization is an effective strategy for improving both potency and stability of linear antimicrobial peptides.

Role of α-Synuclein Regions in Nucleation and Elongation of Amyloid Fiber Assembly.

α-Synuclein is an intrinsically disordered protein whose aggregation in the form of amyloid fibers is directly implicated in Parkinson's disease and other neurological disorders. α-Synuclein is composed of three different regions. The central region (61-95), called NAC, is responsible for protein fibrillation. The N-terminal region (1-61) has some helical propensity and can be divided into H1 (1-31) and H2 (32-61), while the highly acidic C-terminal region (96-140) is completely disordered. It has been postulated that the acidic character of the C-terminus, as well as the interaction between the soluble N- and C- terminal parts, protects the NAC region from fibrillation. In consequence, N- and C-terminal deletions increase α-synuclein fibrillation. Both N- and C-terminal truncations are common in synucleinopathies, but despite their clinical relevance, to date, there are no systematic and exhaustive studies that quantify the effect of these truncations in fiber nucleation and elongation. In this work, we measured both nucleation and fibrillation elongation kinetics in order to study the influence of N- and C-terminal deletions, including the simultaneous deletion of several regions, in α-synuclein fibrillation. We also tested whether the fibrillation prone mutation A53T had an additional effect when combined with truncations. Furthermore, our cross-seeding experiments showed that the deletions studied induce changes in fiber morphology. Our results unravel then the role of the different α-synuclein regions and the A53T mutation in the nucleation and elongation of amyloid fibers.

Secondary Structure Prediction for Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs), without well-defined three-dimensional structures, play vital roles in various biological processes, e.g., protein-/nucleic acid-binding, cellular signaling, and protein regulation. One such role of IDPs/IDRs is the intracellular compartmentalization by forming membraneless organelles via liquid-liquid phase separation (LLPS). These phase-separated protein assemblies are also vital in the design of biomimetic protein-based materials. We recently developed a residue-level coarse-grained (CG) model that has yielded valuable information regarding LLPS of various IDPs [1-2]. However, the current model treats protein chains as flexible molecules in which harmonic bonds link amino acid interaction sites. The model lacks chain rigidity and the ability to populate common secondary structure elements due to the lack of angle and dihedral backbone potentials. Here, we present a new CG model which includes these additional backbone potentials to help capture the transient secondary structures of IDPs. The parameters for the pseudo backbone dihedral potential are transferable and obtained from the context-independent helical propensities of twenty naturally occurring amino acids using NMR [3]. We show that the new model can accurately predict the regions of high helical propensities of IDPs such as TDP-43 as well as PPII propensities of proline-rich regions when compared to NMR and all-atom simulation data. Looking forward, this model can be an essential tool in understanding the sequence-specific LLPS of IDPs and high-throughput sequence protein design. 1. G. L. Dignon, W. Zheng, R. B. Best, Y. C. Kim, and J. Mittal, Proc. Nat. Acad. Sci. 115, 9929-9934, 2018. 2. G. L. Dignon, W. Zheng, Y. C. Kim, and J. Mittal, ACS Cent. Sci. 5, 821-830, 2019. 3. R. J. Moreau, et al., J. Am. Chem. Soc. 131, 13107-13116, 2009.

Strengthening Peptoid Helicity through Sequence Site-Specific Positioning of Amide cis-Inducing NtBu Monomers.

The synthesis of biomimetic helical secondary structures is sought after for the construction of innovative nanomaterials and applications in medicinal chemistry such as the development of protein-protein interaction modulators. Peptoids, a sequence-defined family of oligomers, enable a peptidomimetic strategy, especially considering the easily accessible monomer diversity and peptoid helical folding propensity. However, cis-trans isomerization of the backbone tertiary amides may impair the peptoid's adoption of stable secondary structures, notably the all-cis polyproline I-like helical conformation. Here, we show that cis-inducing NtBu achiral monomers strategically positioned within chiral sequences may reinforce the degree of peptoid helicity, although with a reduced content of chiral side chains. The design principles presented here will undoubtedly help achieve more conformationally stable helical peptoids with desired functions.

Helix breaking transition in the S4 of HCN channel is critical for hyperpolarization-dependent gating.

In contrast to most voltage-gated ion channels, hyperpolarization- and cAMP gated (HCN) ion channels open on hyperpolarization. Structure-function studies show that the voltage-sensor of HCN channels are unique but the mechanisms that determine gating polarity remain poorly understood. All-atom molecular dynamics simulations (~20 μs) of HCN1 channel under hyperpolarization reveals an initial downward movement of the S4 voltage-sensor but following the transfer of last gating charge, the S4 breaks into two sub-helices with the lower sub-helix becoming parallel to the membrane. Functional studies on bipolar channels show that the gating polarity strongly correlates with helical turn propensity of the substituents at the breakpoint. Remarkably, in a proto-HCN background, the replacement of breakpoint serine with a bulky hydrophobic amino acid is sufficient to completely flip the gating polarity from inward to outward-rectifying. Our studies reveal an unexpected mechanism of inward rectification involving a linker sub-helix emerging from HCN S4 during hyperpolarization.

Analysis of Secondary Structure Biases in Naturally Presented HLA-I Ligands.

Recent clinical developments in antitumor immunotherapy involving T-cell related therapeutics have led to a renewed interest for human leukocyte antigen class I (HLA-I) binding peptides, given their potential use as peptide vaccines. Databases of HLA-I binding peptides hold therefore information on therapeutic targets essential for understanding immunity. In this work, we use in depth and accurate HLA-I peptidomics datasets determined by mass-spectrometry (MS) and analyze properties of the HLA-I binding peptides with structure-based computational approaches. HLA-I binding peptides are studied grouping all alleles together or in allotype-specific contexts. We capitalize on the increasing number of structurally determined proteins to (1) map the 3D structure of HLA-I binding peptides into the source proteins for analyzing their secondary structure and solvent accessibility in the protein context, and (2) search for potential differences between these properties in HLA-I binding peptides and in a reference dataset of HLA-I motif-like peptides. This is performed by an in-house developed heuristic search that considers peptides across all the human proteome and converges to a collection of peptides that exhibit exactly the same motif as the HLA-I peptides. Our results, based on 9-mers matched to protein 3D structures, clearly show enriched sampling for HLA-I presentation of helical fragments in the source proteins. This enrichment is significant, as compared to 9-mer HLA-I motif-like peptides, and is not entirely explained by the helical propensity of the preferred residues in the HLA-I motifs. We give possible hypothesis for the secondary structure biases observed in HLA-I peptides. This contribution is of potential interest for researchers working in the field of antigen presentation and proteolysis. This knowledge refines the understanding of the rules governing antigen presentation and could be added to the parameters of the current peptide-MHC class I binding predictors to increase their antigen predictive ability.

Retention of Coiled-Coil Dimer Formation in the Absence of Ion Pairing at Positions Flanking the Hydrophobic Core.

Hydrophobic interactions govern how proteins fold and interact with other molecules, but the impact of nearby polar functionality on the effective hydrophobicity of nonpolar surfaces remains unclear. Here we use a common protein quaternary structure motif, the parallel coiled-coil dimer, to ask whether the identity of basic residues (arginine vs lysine; guanidinium vs ammonium) arrayed along one side of the constituent α-helices influences the favorability of dimerization driven by burial of hydrophobic surface on the other side of each helix. Significant sequence redesign was necessary to achieve the desired juxtaposition of nonpolar and cationic functionality, because we needed to eliminate charged side chains from positions flanking the nonpolar helix surface. Natural and designed sequences that form coiled coils are almost universally rich in acidic and basic residues at these flanking positions. Our arginine coiled-coil dimer was moderately more stable than the lysine analogue, which contrasts with behavior previously observed with helical β-amino acid oligomers bearing guanidinium versus ammonium groups. We attribute this backbone-dependent difference to variations in the extent to which the helical propensities of α- and β-residues can be modulated by design. These findings highlight the challenge of identifying noncovalent forces that direct structure formed by a flexible backbone.

Folding perspectives of an intrinsically disordered transactivation domain and its single mutation breaking the folding propensity

Transcriptional regulation is a critical facet of cellular development controlled by numerous transcription factors, among which are E-proteins (E2A, HEB, and E2-2) that play important roles in lymphopoiesis. For example, primary hematopoietic cells immortalisation is promoted by interaction of the conserved PCET motif consisting of the Leu-X-X-Leu-Leu (LXXLL) and Leu-Asp-Phe-Ser (LDFS) sequences of the transactivation domains (AD1) of E-proteins with the KIX domain of CBP/p300 transcriptional co-activators. Earlier, it was shown that the LXXLL motif is essential for the PCET-KIX interaction driven by the PCET helical transition. In this study, we analyzed the dehydration-driven gain of helicity in the conserved region (residues 11–28) of the AD1 domain of E-protein. Particularly, we showed that AD1 structure was dramatically affected by alcohols, but was insensitive to changes in pH or the presence of osmolytes sarcosine and taurine, or high polyethylene glycol (PEG) concentrations and DOPC Liposomes. These structure-forming effects of solvents were almost completely absent in the case of L21P AD1 mutant characterized by weakened interaction with KIX. This indicates that KIX interaction-induced AD1 ordering is driven by PCET motif dehydration. The L21P mutation-caused loss of molecular recognition function of AD1 is due to the mutation-induced disruption of the AD1 helical propensity.

Ff19SB: Amino-Acid-Specific Protein Backbone Parameters Trained against Quantum Mechanics Energy Surfaces in Solution.

Molecular dynamics (MD) simulations have become increasingly popular in studying the motions and functions of biomolecules. The accuracy of the simulation, however, is highly determined by the molecular mechanics (MM) force field (FF), a set of functions with adjustable parameters to compute the potential energies from atomic positions. However, the overall quality of the FF, such as our previously published ff99SB and ff14SB, can be limited by assumptions that were made years ago. In the updated model presented here (ff19SB), we have significantly improved the backbone profiles for all 20 amino acids. We fit coupled φ/ψ parameters using 2D φ/ψ conformational scans for multiple amino acids, using as reference data the entire 2D quantum mechanics (QM) energy surface. We address the polarization inconsistency during dihedral parameter fitting by using both QM and MM in aqueous solution. Finally, we examine possible dependency of the backbone fitting on side chain rotamer. To extensively validate ff19SB parameters, and to compare to results using other Amber models, we have performed a total of ∼5 ms MD simulations in explicit solvent. Our results show that after amino-acid-specific training against QM data with solvent polarization, ff19SB not only reproduces the differences in amino-acid-specific Protein Data Bank (PDB) Ramachandran maps better but also shows significantly improved capability to differentiate amino-acid-dependent properties such as helical propensities. We also conclude that an inherent underestimation of helicity is present in ff14SB, which is (inexactly) compensated for by an increase in helical content driven by the TIP3P bias toward overly compact structures. In summary, ff19SB, when combined with a more accurate water model such as OPC, should have better predictive power for modeling sequence-specific behavior, protein mutations, and also rational protein design. Of the explicit water models tested here, we recommend use of OPC with ff19SB.

Therapeutic Potential of Trp-Rich Engineered Amphiphiles by Single Hydrophobic Amino Acid End-Tagging.

End-tagging with a single hydrophobic residue contributes to improve the cell selectivity of antimicrobial peptides (AMPs), but systematic studies have been lacking. Thus, this study aimed to systematically investigate how end-tagging with hydrophobic residues at the C-terminus and Gly capped at the N-terminus of W4 (RWRWWWRWR) affects the bioactivity of W4 variants. Among all the hydrophobic residues, only Ala end-tagging improved the antibacterial activity of W4. Meanwhile, Gly capped at the N-terminus could promote the helical propensity of the end-tagged peptides in dodecylphosphocholine micelles, increasing their antimicrobial activities. Of these peptides, GW4A (GRWRWWWRWRA) showed the best antibacterial activity against the 19 species of bacteria tested (GMMIC = 1.86 μM) with low toxicity, thus possessing the highest cell selectivity (TIall = 137.63). It also had rapid sterilization, good salt and serum resistance, and LPS-neutralizing activity. Antibacterial mechanism studies showed that the short peptide GW4A killed bacteria by destroying cell membrane integrity and causing cytoplasmic leakage. Overall, these findings suggested that systematic studies on terminal modifications promoted the development of peptide design theory and provided a potential method for optimization of effective AMPs.

Helix Breaking Transition in the S4 of HCN Channel is Critical for Hyperpolarization-Dependent Gating

HCN channels, unlike other members of the VGIC superfamily, open upon hyperpolarization rather than depolarization. Structural and functional analyses reveal that the voltage-sensor of these channels are unique but the mechanisms that lead to inward rectification remain poorly understood. Here, using all-atom molecular dynamics simulations under hyperpolarization conditions, we report that the S4 voltage-sensor of HCN channels initially moves downward but, upon transfer of the last gating charge, breaks into two sub-helices with the lower sub-helix becoming parallel to the membrane plane. Substitution of the serine at the breakpoint with a hydrophobic residue favors outward rectification but this effect can be reversed by inserting a serine in an adjacent position. We find that the gating polarity strongly correlates with hydrophilicity and helical turn propensity of the substituents at the breakpoint. Our studies reveal an unexpected mechanism of inward rectification involving a linker sub-helix emerging from HCN S4 during hyperpolarization.

Structural characterization and biological activity of Crabrolin peptide isoforms with different positive charge

The search of antimicrobial peptides (AMP) as candidates for the development of antibiotics is an active research field.In this paper we investigated the role of charged residues in antimicrobial activity by using as a template the previously characterized crabrolin peptide.Mutant peptides in which the charge was diminished (Crabrolin Minus) or increased (Crabrolin Plus) were assayed for their ability to inhibit bacterial growth and to bind model bacterial membranes or lipopolysaccharide (LPS). Structural analysis of both peptides by means of CD, NMR and Molecular Dynamics was also performed and correlated to the biological data.Although native Crabrolin (WT) displays smaller efficacy than other antibacterial peptides with similar length, Crabrolin Plus displays a significant antimicrobial activity while Crabrolin Minus is not active, thus confirming the key role of the positive charge for interacting with the bacterial membrane.Moreover, our results show that charge position has no effect on the helical propensity of the peptides but drastically affects their antimicrobial activity.Antimicrobial activity versus Gram-positive and Gram-negative bacteria, as well as specific interaction with LPS, suggest multiple binding modes for the active peptide.

Crystal Structures and Nuclear Magnetic Resonance Studies of the Apo Form of the c-MYC:MAX bHLHZip Complex Reveal a Helical Basic Region in the Absence of DNA.

The c-MYC transcription factor is a master regulator of cell growth and proliferation and is an established target for cancer therapy. This basic helix–loop–helix Zip protein forms a heterodimer with its obligatory partner MAX, which binds to DNA via the basic region. Considerable research efforts are focused on targeting the heterodimerization interface and the interaction of the complex with DNA. The only available crystal structure is that of a c-MYC:MAX complex artificially tethered by an engineered disulfide linker and prebound to DNA. We have carried out a detailed structural analysis of the apo form of the c-MYC:MAX complex, with no artificial linker, both in solution using nuclear magnetic resonance (NMR) spectroscopy and by X-ray crystallography. We have obtained crystal structures in three different crystal forms, with resolutions between 1.35 and 2.2 Å, that show extensive helical structure in the basic region. Determination of the α-helical propensity using NMR chemical shift analysis shows that the basic region of c-MYC and, to a lesser extent, that of MAX populate helical conformations. We have also assigned the NMR spectra of the c-MYC basic helix–loop–helix Zip motif in the absence of MAX and showed that the basic region has an intrinsic helical propensity even in the absence of its dimerization partner. The presence of helical structure in the basic regions in the absence of DNA suggests that the molecular recognition occurs via a conformational selection rather than an induced fit. Our work provides both insight into the mechanism of DNA binding and structural information to aid in the development of MYC inhibitors.