Jungwon Choi

Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP.

Rapamycin, a potent immunosuppressive agent, binds two proteins: the FK506-binding protein (FKBP12) and the FKBP-rapamycin-associated protein (FRAP). A crystal structure of the ternary complex of human FKBP12, rapamycin, and the FKBP12-rapamycin-binding (FRB) domain of human FRAP at a resolution of 2.7 angstroms revealed the two proteins bound together as a result of the ability of rapamycin to occupy two different hydrophobic binding pockets simultaneously. The structure shows extensive interactions between rapamycin and both proteins, but fewer interactions between the proteins. The structure of the FRB domain of FRAP clarifies both rapamycin-independent and -dependent effects observed for mutants of FRAP and its homologs in the family of proteins related to the ataxia-telangiectasia mutant gene product, and it illustrates how a small cell-permeable molecule can mediate protein dimerization.

Refined structure of the FKBP12–rapamycin–FRB ternary complex at 2.2 Å resolution

The structure of the FKBP12-rapamycin-FRB ternary complex has now been refined at 2.2 A resolution. The cell-cycle arrest agent rapamycin binds FK506-binding protein (FKBP12) and the FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin associated protein (FRAP) simultaneously, and the inhibition of FRAP is responsible for rapamycins biological activity. The conformation of rapamycin in the ternary complex is very similar to that observed in the FKBP12-rapamycin binary complex, with an r.m.s. difference of only 0.30 A. However, a slight (9 degrees ) rotation repositions the FRB-binding face of rapamycin in the ternary complex. There are extensive rapamycin-protein interactions and relatively few interactions between the two protein partners FKBP12 and FRB, these interactions mainly involving residues in the 40s and 80s loops of FKBP12 and alpha1 and alpha4 of FRB. The high-resolution refinement has revealed the crucial role of several buried waters in the formation of the ternary complex.

Structure-activity studies of rapamycin analogs: evidence that the C-7 methoxy group is part of the effector domain and positioned at the FKBP12-FRAP interface

BACKGROUND Rapamycin is an immunosuppressant natural product, which blocks T-cell mitogenesis and yeast proliferation. In the cytoplasm, rapamycin binds to the immunophilin FKBP12 and the complex of these two molecules binds to a recently discovered protein, FRAP. The rapamycin molecule has two functional domains, defined by their interaction with FKBP12 (binding domain) or with FRAP (effector domain). We previously showed that the allylic methoxy group at C-7 of rapamycin could be replaced by a variety of different substituents. We set out to examine the effects of such substitutions on FKBP12 binding and on biological activity. RESULTS Rapamycin C-7-modified analogs of both R and S configurations were shown to have high affinities for FKBP12, yet these congeners displayed a wide range of potencies in splenocyte and yeast proliferation assays. The X-ray crystal structures of four rapamycin analogs in complexes with FKBP12 were determined and revealed that protein and ligand backbone conformations were essentially the same as those observed for the parent rapamycin-FKBP12 complex and that the C-7 group remained exposed to solvent. We then prepared a rapamycin analog with a photoreactive functionality as part of the C-7 substituent. This compound specifically labeled, in an FKBP12-dependent manner, a protein of approximately 250 kDa, which comigrates with recombinant FRAP. CONCLUSIONS We conclude that the C-7 methoxy group of rapamycin is part of the effector domain. In the ternary complex, this group is situated in close proximity to FRAP, at the interface between FRAP and FKBP12.

Crystal structure of the C107S/C112S mutant of yeast nuclear 2‐Cys peroxiredoxin

Introduction. Peroxiredoxins (Prxs) are a superfamily of antioxidant enzymes, which are abundant in several isoforms in all kingdoms. Prxs catalyze the reduction of deleterious substances such as hydrogen peroxide (H2O2), alkyl hydroperoxides, and peroxynitrites by utilizing the thiol group of the “peroxidatic” cysteine (CP), which is conserved within the N-terminal region. Some eukaryotic Prxs also act as regulators of H2O2-mediated signal transduction. All Prxs belonging to the thioredoxin-fold superfamily share the same peroxidatic active-site structure. During a catalytic cycle, the CP residue is oxidized by peroxides to a cysteine sulfenic acid (CP-SOH) intermediate. Prxs are classified into 1-Cys and 2-Cys type based on the occurrence of the “resolving” Cys (CR) residue. The 1-Cys Prxs do not contain a CR residue, and the CP-SOH is recycled by glutathionylation mediated by glutathione S-transferase , followed by spontaneous reduction of the enzyme with glutathione. In 2-Cys Prxs, the CP-SOH and CR-SH react to form a stable disulfide, which is then reduced by oxidoreductases such as thioredoxin, tryparedoxin, AhpD, or AhpF. The 2-Cys Prxs have been further subdivided into “typical” and “atypical” types, depending on the position of the CR residue. In typical 2-Cys Prxs (hereafter referred to as T2-Cys Prxs), the CR residue is located within the C-terminal arm of another subunit of a homodimer. In contrast, the CR residue in an atypical 2-Cys Prx resides within the same subunit. The atypical 2-Cys Prxs have also been further subdivided into “L,” “C,” and “R” type subfamilies (hereafter referred to as L-, C-, and R2-Cys Prxs, respectively), depending on the spatial location of the CR residue. 6 Therefore, from a mechanistic point of view, there are five unique Prx subfamilies in total. To date, five distinct Prxs have been identified in the yeast Saccharomyces cerevisiae. They include three thiol peroxidases (cTPx I, II, and III) localized in the cytoplasm, one (nTPx) in the nucleus and one (mTPx) in the mitochondria. cTPx I, II, and III are T2-Cys Prxs, while mTPx is a 1-Cys Prx. nTPx is a member of the C2-Cys Prxs that contains a CxxxxC motif. Bacterial homologues of such Prxs are frequently referred to as the bacterioferritin comigratory proteins (BCP) and their plant homologues are named as PrxQ. These Prxs are least characterized among the Prx subfamilies and information regarding their structure is not yet available. In this study, we have determined the crystal structure of a truncated mutant of nTPx in which both the catalytic residues of Cys107 and Cys112 were replaced with serine. This mutant protein was gradually and spontaneously degraded by the freezing and thawing process until 56 amino acid residues were cleaved off from its N-terminal. nTPx has nuclear targeting sequences but the cleavage site has not yet been determined. The truncated mutant nTPx (hereafter referred to as tmTPx) may correspond to a physiologically mature nTPx. The present structure of a C2-Cys Prx makes it possible to compare the 3D structures of all the five Prx subfamilies.

Crystallization and preliminary X-ray analysis of a truncated mutant of yeast nuclear thiol peroxidase, a novel atypical 2-Cys peroxiredoxin

Saccharomyces cerevisiae nTPx is a thioredoxin-dependent thiol peroxidase that is localized in the nucleus. nTPx belongs to the C-type atypical 2-Cys peroxiredoxin family members, which are frequently called BCPs or PrxQs. A double mutant (C107S/C112S) of nTPx overexpressed in Escherichia coli was spontaneously degraded upon freezing and thawing and its truncated form (residues 57-215; MW = 17837 Da) was crystallized with PEG 3350 and mercury(II) acetate as precipitants using the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.8 A resolution using X-ray synchrotron radiation. The crystals belong to the trigonal space group P3(2), with unit-cell parameters a = b = 37.54, c = 83.26 A. The asymmetric unit contains one molecule of truncated mutant nTPx, with a corresponding VM of 1.91 A3 Da(-1) and a solvent content of 35.5%.