Reconstitution of a native‐like SH2 domain from disordered peptide fragments examined by multidimensional heteronuclear NMR

Abstract

The N-terminal SH2 domain from the p85␣ subunit of phosphatidylinositol 3Ј kinase is cleaved specifically into 9-and 5-kD fragments by limited proteolytic digestion with trypsin. The noncovalent SH2 domain complex and its constituent tryptic peptides have been investigated using high-resolution heteronuclear magnetic resonance (NMR). These studies have established the viability of the SH2 domain as a fragment complementation system. The individual peptide fragments are predominantly unstructured in solution. In contrast, the noncovalent 9-kD + 5-kD complex shows a native-like 1 H-15 N HSQC spectrum, demonstrating that the two fragments fold into a native-like structure on binding. Chemical shift analysis of the noncovalent complex compared to the native SH2 domain reveals that the highest degree of perturbation in the structure occurs at the cleavage site within a flexible loop and along the hydrophobic interface between the two peptide fragments. Mapping of these chemical shift changes on the structure of the domain reveals changes consistent with the reduction in affinity for the target peptide ligand observed in the noncovalent complex relative to the intact protein. The 5-kD fragment of the homologous Src protein is incapable of structurally complementing the p85 9-kD fragment, either in complex formation or in the context of the full-length protein. These high-resolution structural studies of the SH2 domain fragment complementation features establish the suitability of the system for further protein-folding and design studies.Keywords: Fragment complementation; fragment reconstitution; protein-protein interactions; protein folding; nuclear magnetic resonance spectroscopy; limited proteolysis Supplemental material: See www.proteinscience.org.Certain single-domain proteins show native-like structure and function as noncovalent complexes of their constituent peptide fragments. This complementation of domain fragments has been used to study protein-protein interactions in folding/binding events

A Novel Disulfide Bond in the SH2 Domain of the C-terminal Src Kinase Controls Catalytic Activity

“…The sizeable difference in chemical shift values between the respective resonance sets for individual residues is fairly unusual for SH2 domains and suggests that the chemical environment of these nuclei is likely disposed in two unique structural conformations. [16,17] The greatest chemical shift differences occur for residues directly surrounding Cys122 and Cys164 (>5 ppm in the 15 N dimension and >0.3 ppm in the 1 H dimension). Interestingly, the observed chemical shift heterogeneity is not limited to this local environment, but rather is extended to other regions of the structure.…”

The Native-like Interactions between SNase121 and SNase(111−143) Fragments Induce the Recovery of Their Native-like Structures and the Ability to Degrade DNA

“…As is revealed by the analysis of the experimental data, formation of the stable SNase121:SNase(111−143) noncovalent complex occurred through an early suggested folding-on-binding event . The intrinsic native-like structural propensity of SNase121 and SNase(111−143) plays a role in such a refolding event.…”

“…The recovered native-like structure of the complex showed the ability to inhibit chymotrypsin . 9 and 5 kDa fragments of the N-terminal SH2 domain from the p85α subunit of phosphatidylinositol 3′ kinase can fold into a native-like structure on binding to create a noncovalent complex, which retains limited biological activity and can bind their target peptide , . Recently, it was demonstrated that the AroA enzyme and its glyphosate tolerance activities can be directly reconstituted from two separate inactive AroA fragments under both in vivo and in vitro conditions . 1 Abbreviations: SNase, staphylococcal nuclease; CI-2, chymotrypsin inhibitor-2; CD, circular dichroism; pdTp, thymidine 3′,5′-bisphosphate; TMAO, trimethylamine N -oxide; HSQC, heteronuclear single-quantum correlation; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; PDB, Protein Data Bank …”

“…The recovered native-like structure of the complex showed the ability to inhibit chymotrypsin (7). 9 and 5 kDa fragments of the N-terminal SH2 domain from the p85R subunit of phosphatidylinositol 3 0 kinase can fold into a native-like structure on binding to create a noncovalent complex, which retains limited biological activity and can bind their target peptide (8,9). Recently, it was demonstrated that the AroA enzyme and its glyphosate tolerance activities can be directly reconstituted from two separate inactive AroA fragments under both in vivo and in vitro conditions (10).…”

Folding and Misfolding as a Function of Polypeptide Chain Elongation

“…As seen in Section 2, truncated fragments may bear some residual structure even if they are largely unfolded. On the other hand, no such residual structure has been detected (by CD and NMR) in some N-terminal fragments of the SH2 domain 61 and E. coli thioredoxin. 45,62 Heat capacity measurements highlight the important fact that burial of nonpolar surface may occur even in the absence of any significant degree of ordered secondary or tertiary structure.…”