Toward Structure Determination of Disulfide-Rich Peptides Using Chemical Shift-Based Methods

Abstract

Disulfide-rich peptides are a class of molecules for which NMR spectroscopy has been the primary tool for structural characterization. Here, we explore whether the process can be achieved by using structural information encoded in chemical shifts. We examine (i) a representative set of five cyclic disulfide-rich peptides that have high-resolution NMR and X-ray structures and (ii) a larger set of 100 disulfide-rich peptides from the PDB. Accuracy of the calculated structures was dependent on the methods used for searching through conformational space and for identifying native conformations. Although Hα chemical shifts could be predicted reasonably well using SHIFTX, agreement between predicted and experimental chemical shifts was sufficient for identifying native conformations for only some peptides in the representative set. Combining chemical shift data with the secondary structure information and potential energy calculations improved the ability to identify native conformations. Additional use of sparse distance restraints or homology information to restrict the search space also improved the resolution of the calculated structures. This study demonstrates that abbreviated methods have potential for elucidation of peptide structures to high resolution and further optimization of these methods, e.g., improvement in chemical shift prediction accuracy, will likely help transition these methods into the mainstream of disulfide-rich peptide structural biology.

Random coil shifts of posttranslationally modified amino acids

“…Another example of a prediction tool that uses secondary chemical shift input data is ‘DISH’, a method for predicting the dihedral angles of cystine side chains and disulfide bond conformations (Armstrong et al 2018). Finally methods such as CS-ROSETTA (Shen et al 2008, 2009) utilise chemical shifts for de novo structure determination of proteins, and more specialised approaches focussing on disulfide-rich peptides have also been described recently (Wang and Craik 2019). For proteins containing PTMs, the lack of random coil reference values of posttranslationally modified residues means that predictions for some residues cannot be made, or are compromised by using the random coil shift of the corresponding unmodified residue.…”

Supramolecular Peptide Assemblies as Antimicrobial Scaffolds

“…While these non-natural, linear sequences can be engineered to attack bacterial pathogens, nature appears to prefer cyclic peptide building blocks to construct supramolecular antimicrobials. Exemplifying this are polyphemusin-1 (PM-1) [76][77][78][79] found in horseshoe crab hemocytes, θ-defensin BTD-2 [79][80][81] isolated from baboons and protegrin-1 and -4 (PG-1, -4) [78,79,[82][83][84][85] derived from porcine leukocytes, which all adopt cyclic conformations and β-sheet rich amyloid assemblies that potentiate their antimicrobial activity. PM-1, for instance, displays nanomolar activity towards E. coli (MIC = 30 nM), with broad spectrum effects towards a multitude of other pathogens [76][77][78][79].…”

“…Recent structure-activity studies have linked the β-hairpin secondary structure of PM-1 to its ability to form long-range fibrillar assemblies that mediate receptor-independent mechanisms of bactericidal action [79]. Antimicrobial BTD-2 peptides, which are cyclized via central disulfide linkages, have also been recently observed to self-assemble into anti-parallel oligomers to form asymmetric amyloid-like fibrils [79,80]. Interestingly, the co-assembly of BTD-2 with other fibril-forming sequences, such as the tau-derived AcPHF6 peptide, has been shown to enhance the growth and stability of amyloidogenic assemblies [81].…”

Cyclotide Structures Revealed by NMR, with a Little Help from X‐ray Crystallography

“…B) A typical NMR secondary-shift data set from kalata B1. [34] 6] B) The cystine knot motif found in the first solved NMR structure (PDB ID: 1KAL). [5a] C) The cystine knot motif found in the best highresolution NMR structure (PDB ID: 1NB1).…”

“…Copyright: 1995, American Chemical Society. B) A typical NMR secondary‐shift data set from kalata B1 [34] . C) Ribbon representation of the tertiary structure of kalata B1 (PDB ID: 1NB1).Two β‐strands are shown (from position 19–22 and 25–28), the third β‐strand is often distorted and not detected by molecular modelling software.…”