Structural Basis of the Bivalency of the TRPV1 Agonist DkTx.

Bivalency is a prevalent natural mechanism to enhance receptor avidity. Various two-domain disulfide-rich peptides exhibiting bivalent action have been identified from animal venoms. A unique characteristic of these peptides is that they induce a pharmacological response different from that provoked by any of the constituent domains. The enhanced potency and avidity of such peptides is therefore a consequence of their domain fusion by a peptide linker. The role of the linker itself, beyond conjugation, remains unclear. Here, we investigate how the linker affects the bivalency of the capsaicin receptor (TRPV1) agonist DkTx. We recombinantly produced isotope labelled DkTx using a protein splicing approach, to solve the high-resolution solution structure of DkTx, revealing residual linker order stabilised by linker-domain interactions leading to biased domain orientations. The significance of this was studied using a combination of mutagenesis, spin relaxation studies and electrophysiology measurements. Our results reveal that disrupting the pre-organisation of the domains of DkTx is accompanied by reductions in potency and onset of avidity. Our findings support a model of pre-configured two-domain binding, in favour of the previously suggested sequential binding model. This highlights the significance of ordered elements in linker design and the natural evolution of these in bivalent toxins.

Structural basis for the binding of the cancer targeting scorpion toxin, ClTx, to the vascular endothelia growth factor receptor neuropilin-1.

Chlorotoxin (ClTx) is a 36-residue disulfide-rich peptide isolated from the venom of the scorpion Leiurus quinquestriatus. This peptide has been shown to selectively bind to brain tumours (gliomas), however, with conflicting reports regarding its direct cellular target. Recently, the vascular endothelial growth factor receptor, neuropilin-1 (NRP1) has emerged as a potential target of the peptide. Here, we sought to characterize the details of the binding of ClTx to the b1-domain of NRP1 (NRP1-b1) using solution state nuclear magnetic resonance (NMR) spectroscopy. The 3D structure of the isotope labelled peptide was solved using multidimensional heteronuclear NMR spectroscopy to produce a well-resolved structural ensemble. The structure points to three putative protein-protein interaction interfaces, two basic patches (R14/K15/K23 and R25/K27/R36) and a hydrophobic patch (F6/T7/T8/H10). The NRP1-b1 binding interface of ClTx was elucidated using 15N chemical shift mapping and included the R25/K27/R36 region of the peptide. The thermodynamics of binding was determined using isothermal titration calorimetry (ITC). In both NMR and ITC measurements, the binding was shown to be competitive with a known NRP1-b1 inhibitor. Finally, combining all of this data we generate a model of the ClTx:NRP1-b1 complex. The data shows that the peptide binds to the same region of NRP1 that is used by the SARS-CoV-2 virus for cell entry, however, via a non-canonical binding mode. Our results provide evidence for a non-standard NRP1 binding motif, while also providing a basis for further engineering of ClTx to generate peptides with improved NRP1 binding for future biomedical applications.

Protein structural changes characterized by high-pressure, pulsed field gradient diffusion NMR spectroscopy.

Pulsed-field gradient NMR spectroscopy is widely used to measure the translational diffusion and hydrodynamic radius (Rh) of biomolecules in solution. For unfolded proteins, the Rh provides a sensitive reporter on the ensemble-averaged conformation and the extent of polypeptide chain expansion as a function of added denaturant. Hydrostatic pressure is a convenient and reversible alternative to chemical denaturants for the study of protein folding, and enables NMR measurements to be performed on a single sample. While the impact of pressure on the viscosity of water is well known, and our water diffusivity measurements agree closely with theoretical expectations, we find that elevated pressures increase the Rh of dioxane and other small molecules by amounts that correlate with their hydrophobicity, with parallel increases in rotational friction indicated by 13C longitudinal relaxation times. These data point to a tighter coupling with water for hydrophobic surfaces at elevated pressures. Translational diffusion measurement of the unfolded state of a pressure-sensitized ubiquitin mutant (VA2-ubiquitin) as a function of hydrostatic pressure or urea concentration shows that Rh values of both the folded and the unfolded states remain nearly invariant. At ca 23 Å, the Rh of the fully pressure-denatured state is essentially indistinguishable from the urea-denatured state, and close to the value expected for an idealized random coil of 76 residues. The intrinsically disordered protein (IDP) α-synuclein shows slight compaction at pressures above 2 kbar. Diffusion of unfolded ubiquitin and α-synuclein is significantly impacted by sample concentration, indicating that quantitative measurements need to be carried out under dilute conditions.

Observation and Kinetic Characterization of Transient Schiff Base Intermediates by CEST NMR Spectroscopy.

In aqueous solution, many biochemical reaction pathways involve reaction of an aldehyde with an amine, which progresses through generally unstable, hydrated and dehydrated, Schiff base intermediates that often are unobservable by conventional NMR. There are 4 states in the relevant equilibrium: 1) gem-diol, 2) aldehyde, 3) hemiaminal, and 4) Schiff base. For the reaction between protein amino groups and DOPAL, a highly toxic metabolite of dopamine, the 1 H resonances of both the hemiaminal and the dehydrated Schiff base can be observed by CEST NMR, even when their populations fall below 0.1 %. CEST NMR reveals the quantitative exchange kinetics between reactants and Schiff base intermediates, explaining why the Schiff base NMR signals are rarely observed. The reactivity of DOPAL with Nα -amino groups is greater than with lysine Nϵ -amines and, in the presence of O2 , both types of Schiff base DOPAL-peptide intermediates rapidly react with free DOPAL to irreversibly form dicatechol pyrrole adducts.

Residual Dipolar Couplings for Resolving Cysteine Bridges in Disulfide-Rich Peptides.

Disulfide bridges in proteins are formed by the oxidation of pairs of cysteine residues. These cross-links play a critical role in stabilizing the 3D-structure of small disulfide rich polypeptides such as hormones and venom toxins. The arrangement of the multiple disulfide bonds directs the peptide fold into distinct structural motifs that have evolved for resistance against biochemical and physical insults. These structural scaffolds have, therefore, proven to be very attractive in bioengineering efforts to develop novel biologics with applications in health and agriculture. Structural characterization of small disulfide rich peptides (DRPs) presents unique challenges when using commonly applied biophysical methods. NMR is the most commonly used method for studying such molecules, where the relatively small size of these molecules results in highly precise structural ensembles defined by a large number of distance and dihedral angle restraints per amino acid. However, in NMR the sulfur atoms that are involved in three of the five dihedral angles in a disulfide bond cannot be readily measured. Given the central role of disulfide bonds in the structure of these molecules, it is unclear what the inherent resolution of such NMR structures is when using traditional NMR methods. Here, we use an extensive set of long-range residual dipolar couplings (RDCs) to assess the resolution of the NMR structure of a disulfide-rich peptide. We find that structures based primarily on NOEs, yield ensembles that are equivalent to a crystallographic resolution of 2-3 Å in resolution, and that incorporation of RDCs reduces this to ~1-1.5 Å resolution. At this resolution the sidechain of ordered amino acids can be defined accurately, allowing the geometry of the cysteine bridges to be better defined, and allowing for disulfide-bond connectivities to be determined with high confidence. The observed improvements in resolution when using RDCs is remarkable considering the small size of these peptides.

The structure, dynamics and selectivity profile of a NaV1.7 potency-optimised huwentoxin-IV variant.

Venom-derived peptides have attracted much attention as potential lead molecules for pharmaceutical development. A well-known example is Huwentoxin-IV (HwTx-IV), a peptide toxin isolated from the venom of the Chinese bird-eating spider Haplopelma schmitdi. HwTx-IV was identified as a potent blocker of a human voltage-gated sodium channel (hNaV1.7), which is a genetically validated analgesic target. The peptide was promising as it showed high potency at NaV1.7 (IC50 ~26 nM) and selectivity over the cardiac NaV subtype (NaV1.5). Mutagenesis studies aimed at optimising the potency of the peptide resulted in the development of a triple-mutant of HwTx-IV (E1G, E4G, Y33W, m3-HwTx-IV) with significantly increased potency against hNaV1.7 (IC50 = 0.4 ± 0.1 nM) without increased potency against hNaV1.5. The activity of m3-HwTx-IV against other NaV subtypes was, however, not investigated. Similarly, the structure of the mutant peptide was not characterised, limiting the interpretation of the observed increase in potency. In this study we produced isotope-labelled recombinant m3-HwTx-IV in E. coli, which enabled us to characterise the atomic-resolution structure and dynamics of the peptide by NMR spectroscopy. The results show that the structure of the peptide is not perturbed by the mutations, whilst the relaxation studies reveal that residues in the active site of the peptide undergo conformational exchange. Additionally, the NaV subtype selectivity of the recombinant peptide was characterised, revealing potent inhibition of neuronal NaV subtypes 1.1, 1.2, 1.3, 1.6 and 1.7. In parallel to the in vitro studies, we investigated NaV1.7 target engagement of the peptide in vivo using a rodent pain model, where m3-HwTx-IV dose-dependently suppressed spontaneous pain induced by the NaV1.7 activator OD1. Thus, our results provide further insight into the structure and dynamics of this class of peptides that may prove useful in guiding the development of inhibitors with improved selectivity for analgesic NaV subtypes.

The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large ß-hairpin loop.

Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Sf1a, a 46-residue peptide isolated from the venom of the tube-web spider Segesteria florentina, is insecticidal to a wide range of insects, but nontoxic to vertebrates. In order to investigate its structure and mode of action, we developed an efficient bacterial expression system for the production of Sf1a. We determined a high-resolution solution structure of Sf1a using multidimensional 3D/4D NMR spectroscopy. This revealed that Sf1a is a knottin peptide with an unusually large ß-hairpin loop that accounts for a third of the peptide length. This loop is delimited by a fourth disulfide bond that is not commonly found in knottin peptides. We showed, through mutagenesis, that this large loop is functionally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its 'depressant' paralytic phenotype in insects. However, in contrast to the majority of spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to act as a pore blocker.

Ca2+ binding enhanced mechanical stability of an archaeal crystallin.

Structural topology plays an important role in protein mechanical stability. Proteins with ß-sandwich topology consisting of Greek key structural motifs, for example, I27 of muscle titin and (10)FNIII of fibronectin, are mechanically resistant as shown by single-molecule force spectroscopy (SMFS). In proteins with ß-sandwich topology, if the terminal strands are directly connected by backbone H-bonding then this geometry can serve as a "mechanical clamp". Proteins with this geometry are shown to have very high unfolding forces. Here, we set out to explore the mechanical properties of a protein, M-crystallin, which belongs to ß-sandwich topology consisting of Greek key motifs but its overall structure lacks the "mechanical clamp" geometry at the termini. M-crystallin is a Ca(2+) binding protein from Methanosarcina acetivorans that is evolutionarily related to the vertebrate eye lens ß and γ-crystallins. We constructed an octamer of crystallin, (M-crystallin)8, and using SMFS, we show that M-crystallin unfolds in a two-state manner with an unfolding force ∼ 90 pN (at a pulling speed of 1000 nm/sec), which is much lower than that of I27. Our study highlights that the ß-sandwich topology proteins with a different strand-connectivity than that of I27 and (10)FNIII, as well as lacking "mechanical clamp" geometry, can be mechanically resistant. Furthermore, Ca(2+) binding not only stabilizes M-crystallin by 11.4 kcal/mol but also increases its unfolding force by ∼ 35 pN at the same pulling speed. The differences in the mechanical properties of apo and holo M-crystallins are further characterized using pulling speed dependent measurements and they show that Ca(2+) binding reduces the unfolding potential width from 0.55 nm to 0.38 nm. These results are explained using a simple two-state unfolding energy landscape.

Conformational propensities and dynamics of a ßγ-crystallin, an intrinsically disordered protein.

The three-dimensional folded structure of a protein has been considered essential for its function. However, recently many proteins have been identified to function without having a definite structure and they have been classified as intrinsically disordered proteins (IDPs). Recently, we have identified a ßγ-crystallin domain in the genome of a marine bacterium called Hahella chejuensis on the basis of known sequence signatures. This protein, called Hahellin, was characterized by NMR spectroscopy as an IDP, which upon Ca(2+)-binding was shown to undergo a large conformational transformation and acquires a typical ßγ-crystallin fold. In this paper, we have characterized this IDP by a combined use of NMR and Replica Exchange Molecular Dynamics simulation and found it to be in a highly dynamic, inter-converting population having a molten globular state with the C-terminal Greek key motif relatively more flexible as compared to its N-terminal counterpart. Network analysis and clustering on the observed conformational ensemble showed a heterogeneous mixture of eleven distinct clusters, classified into near-native and far-native populations, which are not in equilibrium. Several conformational clusters display an increased propensity for helical conformation(s) and a decreased ß-strand propensity, which is consistent with the NMR observations made on this protein. The negatively charged Ca(2+)-coordinating residues form parts of the highly flexible polypeptide stretches, and thus act as seeds for the origin of different conformational clusters observed. This study thus helps us to understand the relationship between the role of conformational dynamics and the structural propensities of the intrinsically disordered state of apo-Hahellin.

Backbone ¹H, ¹³C and ¹5N resonance assignments of an intrinsically unstructured ßγ-crystallin from Hahella chejuensis.

The sequence specific backbone (1)H, (13)C and (15)N resonance assignments of an intrinsically unstructured ßγ-crystallin from Hahella chejuensis are reported. The secondary structure chracterization of the unstructured protein reveals that large fraction of residues exhibits ß-strand propensity, as in the case of the Ca(2+)-bound structured protein.

Iterative cloning, overexpression, purification and isotopic labeling of an engineered dimer of a Ca(2+)-binding protein of the ßγ-crystallin superfamily from Methanosarcina acetivorans.

ßγ-Crystallins are a large superfamily of proteins found in vertebrate eye lens. They are hetero-dimers (linked in tandem by a specific peptide) and are shown to bind calcium. The monomers possess two ß-strand rich greek-key motifs. Recently, a structurally closest member to the family of lens ßγ-crystallins has been described, for the first time, from the archaea Methanosarcina acetivorans, which is named as M-crystallin. Unlike lens ßγ-crystallins, M-crystallin exits as a monomer. Here, we synthesized a dimeric gene of M-crystallin in which two monomers are linked by a 10-amino acid residue coding sequence. The linker sequence in the target protein is long and flexible enough to reduce the proximity between the individual crystallins in the dimer. This methodology would be highly beneficial in designing polyproteins (two or more proteins linked in tandem to aid mechanical stretching studies) that are regularly used in single-molecule force spectroscopy. The dimer of M-crystallin was overexpressed in Escherichia coli BLR(DE3) strain. The overexpressed protein containing an N-terminal hexa-histidine tag was purified using nickel affinity chromatography and then by size-exclusion chromatography. Further, a method to purify isotopically ((15)N) labeled protein with high yield for NMR studies is reported. The uniformly (15)N-labeled M-crystallin dimer thus produced has been characterized by recording sensitivity enhanced 2D [(15)N-(1)H] HSQC and other optical spectroscopy techniques. Observation of only one set of peaks in the HSQC, along with the structural characterization using optical spectroscopy, suggests that the domains in the dimer possess similar structure as that of the monomer.