Analysis of disulfide bonds in protein structures.

Abstract

Disulfide bonds are covalent links between the sulfur atoms of two cysteine residues in the polypeptide chain. To assist with identification of possible functional disulfide bonds in proteins, we have generated a simple interface to obtain relevant information about disulfide bonds in crystal or solution structures. This includes secondary structural information, solvent accessibility values and different geometric measures. Most disulfide bonds are structural motifs that assist protein folding and stabilize the mature form. Some disulfide bonds serve a functional role and have been classified as either catalytic or allosteric bonds. The catalytic bonds are found in the active sites of oxidoreductases, proteins that mediate thiol/disulfide exchange in other proteins. The allosteric bonds are defined as bonds that control the function of the protein in which they reside by mediating a change when they break (reduce) or form (oxidize). The redox state of the allosteric disulfides are controlled by the catalytic disulfides. A disulfide bond is defined by six atoms of the two cysteine residues (Cα–Cβ–Sγ–Sγ′–Cβ′–Cα’) and five chi (χ) angles, which are the rotation about the bonds linking the atoms. There are 20 possible disulfide bond configurations based on the sign of each chi angle. For example, a disulfide is a positive left handed spiral (+LHSpiral) if the χ1, χ2, χ3, χ2′ and χ1′ angles are positive, positive, negative, positive and negative, respectively. The functional disulfides have been associated with particular conformations. The catalytic disulfides are nearly always ±RHHooks because they occur in a common secondary structure. The allosteric disulfides will have different conformations, although most allosteric bonds identified so far have a –RHStaple configuration and link β‐strands or β‐loops. Specific structural information from PDB structures is generally difficult to extract without expert knowledge in the scripting of 3D structure viewing and manipulation programs such as PyMol, Swiss‐PDB and RasMol. In particular, there are often multiple disulfide bonds within a single structure making it a formidable task in extracting geometric information. The issue is exacerbated for NMR structures where it is often useful to analyze the disulfide conformations within each model separately. We have developed a simple interface for researchers interested in obtaining information about disulfide bonds within any PDB structure: http://www.cancerresearch.unsw.edu.au/CRCWeb.nsf/page/Disulfide+Bond+Analysis. The software, Disulfide Bond Analysis, can be accessed through any modern web browser. It is able to accept any file that complies with the PDB format (.pdb or.ent). Alternatively, a user may input a list of PDB IDs that are already present in the RSCB protein data bank. Each PDB file is analyzed using DSSP to generate secondary structure characteristics and solvent accessibility values of the disulfide bond cysteines. Subsequently, the program computes the chi angles of the disulfide bond, calculates the dihedral strain energy and assigns the conformation. The distance between the α‐carbon atoms of the disulfide cysteines is also computed as a value < 5 Å is predictive of allosteric –RHStaple bonds. All the information is then compiled and a summary of the results are immediately displayed on the webpage, or the complete analysis can be downloaded in Microsoft® Excel format (.xls). A tutorial describing the use of Disulfide Bond Analysis along with a detailed description of the output can be found at the website. Disulfide Bond Analysis provides a simple web interface for researchers to obtain disulfide bond characteristics from PDB structures. The analysis can be completed in seconds and without detailed knowledge of the PDB file format. The program can be used to identify potential functional disulfides in proteins based on the known features of these bonds. The authors state that they have no conflict of interest. The authors thank B. Schmidt, L. Ho and K. Hu for their contributions to early versions of this program. This study was supported by a grant from the National Health and Medical Research Council of Australia.