Cyclized Peptide Inhibitors of the Small G Protein Cdc42 Mimic Binding of Effector Proteins

TL;DR

Researchers developed cyclized peptides that inhibit Cdc42, a small G protein, by mimicking the binding of natural effector proteins.

Contribution

The study presents a novel structural mechanism for inhibiting Cdc42 using cyclized peptides that mimic effector binding.

Findings

Third-generation cyclized peptides showed improved affinity for Cdc42 through amino acid substitutions.

The peptide binds to Cdc42 in a β-hairpin conformation, extending the β-sheet of the GTPase Rossman fold.

NMR structures of unbound peptide variants informed the rational design of substituted inhibitors.

Abstract

The Ras superfamily of small GTPases are challenging
targets for
therapeutic inhibition, partially due to a lack of pockets amenable
to small molecule inhibition. Our previous work identified high-affinity
cyclized peptide binders of Cdc42, a member of the Rho family of small
GTPases, capable of inhibiting activity. To further optimize these
Cdc42 inhibitors, we have engineered modifications to the best sequence
available from the original maturation and screened the ability of
these third-generation peptides to compete with Cdc42-effector interactions.
Improvements in affinity were achieved by single amino acid substitutions
at several residue positions. We present the structure of one of these
nanomolar affinity, cyclized peptides in complex with Cdc42. The structure
reveals that the peptide binds in a β-hairpin conformation to
create an extension of the β-sheet of the GTPase Rossman fold,
acting as a structural mimic of native Cdc42 effectors. We additionally
elucidate the NMR structures of four unbound C-terminal alanine variants
and employ both the bound and unbound structures to inform the rational
design of substituted peptide inhibitors. Overall, this study expands
our understanding of how Ras GTPases can be targeted, by demonstrating
a rare example of an inhibitor binding contiguously with a surface
of β-strand of the small G protein, which illustrates an innovative
avenue for noncovalent therapeutic design.

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