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

Venkatraman Ramanujam,Yang Shen,Jinfa Ying,Mehdi Mobli

doi:10.3389/fchem.2019.00889

Abstract

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.

Highlights

Disulfide bridges are naturally occurring cross-links formed between the side chains of two cysteine residues and are one of the most important post-translational modifications in proteins
When we compared the alignment of each individual helix from either the published structure or that refined here without residual dipolar couplings (RDCs) we find these to align very well with the RDC refined structure along the backbone (RMSD ∼ 0.2 Å), indicating that the helices are locally accurately defined in all structures
Our analysis suggests that including RDCs in such a data-driven approach provides much higher confidence in determining disulfide bond connectivities and is unlikely to lead to incorrect solutions

Summary

Introduction

Disulfide bridges are naturally occurring cross-links formed between the side chains of two cysteine residues and are one of the most important post-translational modifications in proteins. Disulfide-rich peptides and proteins are commonly secreted, and include biopharmaceutical targets such as hormones and antibodies (Lewis and Garcia, 2003; Mamathambika and Bardwell, 2008; Gongora-Benitez et al, 2014). In these molecules, the disulfide bonds serve to stabilize the protein fold in the extracellular environment. As research efforts in this field intensify there is an interest in defining the high-resolution structure of these proteins to interpret structureactivity relationship studies and for rational peptide engineering (Brust et al, 2013)

Methods

Results

Discussion

Conclusion