A minimalist approach toward protein recognition by epitope transfer from functionally evolved beta-sheet surfaces.
New approaches for identifying small molecules that specifically target protein surfaces as opposed to active site clefts are of much current interest. Toward this goal, we describe a three-step methodology: in step one, we target a protein of interest by directed evolution of a small beta-sheet scaffold; in step two, we identify residues on the scaffold that are implicated in binding; and in step three, we transfer the chemical information from the beta-sheet to a small molecule mimic. As a case study, we targeted the proteolytic enzyme thrombin, involved in blood coagulation, utilizing a library of beta-sheet epitopes displayed on phage that were previously selected for conservation of structure. We found that the thrombin-binding, beta-sheet displaying mini-proteins retained their structure and stability while inhibiting thrombin at low micromolar inhibition constants. A conserved dityrosine recognition motif separated by 9.2 A was found to be common among the mini-protein inhibitors and was further verified by alanine scanning. A molecule containing two tyrosine residues separated by a linker that matched the spacing on the beta-sheet scaffold inhibited thrombin, whereas a similar dityrosine molecule separated by a shorter 6 A linker could not. Moreover, kinetic analysis revealed that both the mini-protein as well as its minimalist mimic with only two functional residues exhibited noncompetitive inhibition of thrombin. Thus, this reductionist approach affords a simple methodology for transferring information from structured protein scaffolds to yield small molecule leads for targeting protein surfaces with novel mechanisms of action.
Rajagopal
Srivats
S
Department of Chemistry, University of Arizona, P. O. Box 210041, 1306 East University Boulevard, Tucson, Arizona 85721-0041, USA.
Meyer
Scott C
SC
Goldman
Aaron
A
Zhou
Min
M
Ghosh
Indraneel
I
D000595:Amino Acid Sequence; D001665:Binding Sites; D000939:Epitopes / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
D000939:Epitopes; D007074:Immunoglobulin G; D011506:Proteins; D014443:Tyrosine; D013917:Thrombin