Abstract
We have investigated covalent conjugation of VPPPVPPRRRX′ peptide (where X′ denotes Nε-chloroacetyl lysine) to N-terminal SH3 domain from adapter protein Grb2. Our experimental results confirmed that the peptide first binds to the SH3 domain noncovalently before establishing a covalent linkage through reaction of X′ with the target cysteine residue C32. We have also confirmed that this reaction involves a thiolate-anion form of C32 and follows the SN2 mechanism. For this system, we have developed a new MD-based protocol to model the formation of covalent conjugate. The simulation starts with the known coordinates of the noncovalent complex. When two reactive groups come into contact during the course of the simulation, the reaction is initiated. The reaction is modeled via gradual interpolation between the two sets of force field parameters that are representative of the noncovalent and covalent complexes. The simulation proceeds smoothly, with no appreciable perturbations to temperature, pressure or volume, and results in a high-quality MD model of the covalent complex. The validity of this model is confirmed using the experimental chemical shift data. The new MD-based approach offers a valuable tool to explore the mechanics of protein-peptide conjugation and build accurate models of covalent complexes.
Highlights
Various natural and modified peptides are broadly used in modern clinical practice
Noncovalent binding between Sos1-X′ and Grb[2] N-SH3
Grb[2] is an adaptor protein that consists of a central SH2 domain, which preferentially binds to phosphotyrosine-containing motifs, and two flanking SH3 domains, which bind to proline-rich motifs
Summary
Various natural and modified peptides are broadly used in modern clinical practice. For example, gramicidin (topical antibiotic), insulin (life-saving diabetes treatment), oxytocin (used to induce and support labor), goserelin and degarelix (prostate cancer drugs) all fall in this category[1]. One interesting direction in this area is development of peptides that are capable of covalently binding to their targets (and belong to a broad class of covalent drugs) This can be accomplished via structure-based design. The peptide needs to be re-designed such as to incorporate a reactive group in the selected position This group should be capable of forming a bond with one of the protein moieties that are located nearby in the structure of the complex. The peptide forms a noncovalent complex with its protein target This brings the peptide’s reactive group in close proximity to its target moiety on the protein surface, achieving high “local concentration of the reactants”. Potential gains from the use of reactive peptides are likely less than can be (naively) expected
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