Conserved solvent and side-chain interactions in the 1.35 Å structure of the Kelch domain of Keap1

Abstract

The Kelch repeat is a common sequence motif in eukaryotic genomes and is approximately 50 amino acids in length. The structure of the Kelch domain of the human Keap1 protein has previously been determined at 1.85 Angstrom, showing that each Kelch repeat forms one blade of a six-bladed beta-propeller. Here, use of 1.35 Angstrom SAD data for de novo structure determination of the Kelch domain and for refinement at atomic resolution is described. The high quality and resolution of the diffraction data and phase information allows a detailed analysis of the role of solvent in the structure of the Kelch repeat. Ten structurally conserved water molecules are identified in each blade of the Kelch beta-propeller. These appear to play distinct structural roles that include lining the central channel of the propeller, interacting with residues in loops between strands of the blade and making contacts with conserved residues in the Kelch repeat. Furthermore, we identify a conserved C-H...pi hydrogen bond between two key residues in the consensus Kelch repeat. This analysis extends our understanding of the structural roles of conserved residues in the Kelch repeat and highlights the potential role of solvent in maintaining the fold of this common eukaryotic structural motif.

Novel β-Propeller of the BTB-Kelch Protein Krp1 Provides a Binding Site for Lasp-1 That Is Necessary for Pseudopodial Extension

“…A detailed examination of the five Krp1 Kelch repeats shows that only two display the full repertoire of the signature motifs. The structure of Keap1 provided the opportunity to assign structural roles to the signature motifs of the Kelch repeat (10,13,14,31), an ϳ50-amino acid polypeptide that folds in a four- Consistent with a Kelch repeat, there are 4 hydrophobic residues on strand B (yellow) and a tyrosine in the correct position in strand C (magenta) of the Krp1 blade I. However, there is no glycine doublet at the end of strand B, and the glycines are replaced by asparagine and aspartic acid (cyan).…”

“…The highest degree of variation in the structure is in the interstrand loops, particularly the loop linking strand B to strand C. c, a standard Kelch arrangement is found in blade III of Krp1 (panel i). The current definition of a Kelch repeat requires 4 hydrophobic amino acids followed by a glycine doublet to form strand B and a tyrosine toward the end of strand C that interacts with a tryptophan on strand D. It has been proposed that the hydrophobic interactions between the aromatic rings of the conserved tyrosine and tryptophan are critical to blade folding and that additional hydrogen bonding from the tyrosine hydroxyl group is also important (31). However, the tyrosine is not completely conserved in Krp1 as this position is occupied by a phenylalanine in blade V, eliminating the availability of a hydroxyl group from hydrogen bonding, and a leucine in blade II, demonstrating that the hydrophobic residue at this position need not be an aromatic (panel ii).…”

Non-electrophilic modulators of the canonical Keap1/Nrf2 pathway

“…41,47,52 The crystal structure showed that the aromatic ring of THIQ settles at the top of the binding pocket with potential space to incorporate new interactions with the pocket: the cyclohexanecarboxylic acid moiety makes interactions with Tyr 572, Ser 602, Arg 415, and Asn 414, and the phthalimide forms hydrogen bonds and a π-stacking complex with Ser 602 and Tyr 572, respectively. The parent compound possesses moderate affinity for Keap1, IC 50 = 2.3 µM with similar potencies seen in reporter gene assays, but linked to the THIQ core is a phthalimide moiety, which was removed by Jnoff et al, 48 presumably because of its poor physicochemical and pharmaceutical properties.…”

Minimizing frustration by folding in an aqueous environment

“…Not all conserved water molecules are buried, although many thought to stabilize significantly the native state are found in the interior of the protein to bridge polar groups that would otherwise be too far apart to interact. The 1.35 Å crystal structure of the Kelch domain of Keap1 provides a prime example of how conserved water molecules participate in maintaining the structural integrity of the protein [116]. The Kelch domain is composed of a six-bladed β-propeller where each blade consists of a four-stranded antiparallel twisted β-sheet containing ten strictly conserved water molecules ( Figure 6).…”

“…The remaining six waters bridge between various highly conserved residues in the loops or strands at the internal parts of the propeller. The strict conservation of the water binding sites reflected in the residues with which they interact, suggest that these water molecules play a prominent role in stabilizing the folded state and are likely to be important in the protein folding process [116].…”