Identification and Rational Redesign of Peptide Ligands to CRIP1, A Novel Biomarker for Cancers

Cysteine-rich intestinal protein 1 (CRIP1) has been identified as a novel marker for early detection of cancers. Here we report on the use of phage display in combination with molecular modeling to identify a high-affinity ligand for CRIP1. Panning experiments using a circularized C7C phage library yielded several consensus sequences with modest binding affinities to purified CRIP1. Two sequence motifs, A1 and B5, having the highest affinities for CRIP1, were chosen for further study. With peptide structure information and the NMR structure of CRIP1, the higher-affinity A1 peptide was computationally redesigned, yielding a novel peptide, A1M, whose affinity was predicted to be much improved. Synthesis of the peptide and saturation and competitive binding studies demonstrated approximately a 10–28-fold improvement in the affinity of A1M compared to that of either A1 or B5 peptide. These techniques have broad application to the design of novel ligand peptides

Diminished Self-Chaperoning Activity of the ΔF508 Mutant of CFTR Results in Protein Misfolding

The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the ΔF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the ΔF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-ΔF508 variants exhibited significantly higher folding probabilities than the original NBD1-ΔF508, thereby partially rescuing folding ability of the NBD1-ΔF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-ΔF508 are essential information in correcting this pathogenic mutant

A) The Phylogenetic tree of cetacean Mb upon the divergence from terrestrial counterparts.

<p>Ancestral states were inferred using the maximum likelihood (ML) approach described in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002929#s3" target="_blank">Methods </a><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002929#pcbi.1002929-Tamura1" target="_blank">[59]</a>. Amino acid changes in each branch are shown with the respective changes in free energy of folding, ΔΔG in kcal/mol calculated from the FoldX force field <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002929#pcbi.1002929-Schymkowitz1" target="_blank">[28]</a>. Stabilization and destabilization is presented by red and blue colors respectively across the phylogeny, with branch height proportional to |ΔΔG| of that specific branch. B) The average ω = dN/dS for the variable sites in A from the M8 model is plotted versus the average ΔΔG of mutations in these sites. C) The distribution of mutational effects in Mb from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002929#pcbi.1002929-Tokuriki1" target="_blank">[36]</a> is shown with the solid black line where arrows show the average ΔΔG for an average mutation in Mb (∼1.22 kcal/mol), in the cetacean clade among not-positively selected mutations (∼0.06 kcal/mol) and, among the positively selected residues (∼−0.26 kcal/mol). The probability of stabilization caused by positive selection is ∼0.8.</p

The Bayes empirical Bayes predictions for ω values for each site in cetacean Mb.

<p>A) For each residue p(ω<1), p(ω = 1) and p(ω>1) are shown in cyan, green and red respectively. Residues 5, 21, 22, 35, 51, 66, 121, and 129 have probabilities (ω>1)>0.5 with <ω> = 5.86 from the M8 model using the ML-estimated branch lengths under the M0 model. B) Crystal structure of sperm whale Mb taken from the protein data bank (ID = 1U7S) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002929#pcbi.1002929-Phillips1" target="_blank">[32]</a> with residues color coded by p(ω). The figure was created using PyMOL (<a href="http://www.pymol.org" target="_blank">http://www.pymol.org</a>).</p