High-Resolution Epitope Mapping of hGH-Receptor Interactions by Alanine-Scanning Mutagenesis

Abstract

A strategy, called alanine-scanning mutagenesis, was used to identify specific side chains in human growth hormone (hGH) that strongly modulate binding to the hGH receptor cloned from human liver. Single alanine mutations (62 in total) were introduced at every residue contained within the three discontinuous segments of hGH (residues 2 to 19, 54 to 74, and 167 to 191) that have been implicated in receptor recognition. The alanine scan revealed a cluster of a dozen large side chains that when mutated to alanine each showed more than a four times lower binding affinity to the hGH receptor. Many of these residues that promote binding to the hGH receptor are altered in homologs of hGH (such as placental lactogens and prolactins) that do not bind tightly to the hGH receptor. The overall folding of these mutant proteins was indistinguishable from that of the wild-type hGH, as determined by strong cross-reactivities with seven different conformationally sensitive monoclonal antibodies. The alanine scan also identified at least one side chain, Glu174, that hindered binding because when it was mutated to alanine the receptor affinity increased by more than a factor of four.

Rational disruption of the oligomerization of the mini‐ferritin E. coli DPS through protein‐protein interface mutation

“…12,13 The protein has three types of protein-protein interfaces that are related to the twofold and the Dps-like and the ferritin-like threefold symmetry axes. The Dps-like threefold interface buries only a small amount of monomer surface area, and salt bridging interactions or aromatic amino acids that are prevalent in hot spots (Trp or Tyr) are not evident in this interface.…”

“…9,10 Alanine shaving, where individual side changes are conceptually shaved to a methyl residuum and where the stabilities of the resulting mutants are determined with respect to wild type, is the most common method to identify hot spot residues. [11][12][13] This study uses alanine shaving to identify hot spots in a mini-ferritin nano-cage protein.…”

Binding of amino acid side chains to preformed cavities: Interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues

“…This method has been greatly extended to protein-protein association by the widely used alanine scan of Cunningham and Wells (1989) where a large set of mutants of one of the partner proteins is prepared, with systematic one-at-a-time replacements of existing residues by Ala. If an Ala replacement causes a big change in binding the position is judged to be a part of the combining region.…”

A conserved region in the σ54‐dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation

“…For these experiments, we used a truncated form of the protein, referred to as DctD (⌬1-142) , which lacks the N-terminal regulatory domain and is constitutively active (Lee et al, 1994). We also performed alanine-scanning mutagenesis (Cunningham and Wells, 1989) on an eight amino acid sequence in the N-terminal half of the C3 region to define the function of this region. Characterization of mutant forms of DctD (⌬1-142) with amino acid substitutions in the C3 region suggested that the N-terminal half of this region contains a site that contacts 54 -holoenzyme.…”