Structure of an Hsp90-immunophilin complex reveals cochaperone recognition of the client-maturation state

SummaryThe Hsp90 chaperone promotes the folding and activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct cochaperone regulators. The FKBP51 immunophilin binds Hsp90 with its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during the folding of kinases, nuclear receptors and tau. Here we have determined the cryo-EM structure of the human Hsp90:FKBP51:p23 complex to 3.3 Å that, together with mutagenesis and crosslinking analysis, reveals the basis for cochaperone binding to Hsp90 during client maturation. A helix extension in the TPR functions as a key recognition element, interacting across the Hsp90 C-terminal dimer interface presented in the closed, ATP conformation. The PPIase domain is positioned along the middle domain, adjacent Hsp90 client binding sites, while a single p23 makes stabilizing interactions with the N-terminal dimer. With this architecture, FKBP51 could thereby act on specific client residues presented during Hsp90-catalyzed remodeling.

Coupled intra- and interdomain dynamics support domain cross-talk in Pin1

The functional mechanisms of multidomain proteins often exploit interdomain interactions, or “cross-talk.” An example is human Pin1, an essential mitotic regulator consisting of a Trp–Trp (WW) domain flexibly tethered to a peptidyl-prolyl isomerase (PPIase) domain, resulting in interdomain interactions important for Pin1 function. Substrate binding to the WW domain alters its transient contacts with the PPIase domain via means that are only partially understood. Accordingly, we have investigated Pin1 interdomain interactions using NMR paramagnetic relaxation enhancement (PRE) and molecular dynamics (MD) simulations. The PREs show that apo-Pin1 samples interdomain contacts beyond the range suggested by previous structural studies. They further show that substrate binding to the WW domain simultaneously alters interdomain separation and the internal conformation of the WW domain. A 4.5-μs all-atom MD simulation of apo-Pin1 suggests that the fluctuations of interdomain distances are correlated with fluctuations of WW domain interresidue contacts involved in substrate binding. Thus, the interdomain/WW domain conformations sampled by apo-Pin1 may already include a range of conformations appropriate for binding Pin1's numerous substrates. The proposed coupling between intra-/interdomain conformational fluctuations is a consequence of the dynamic modular architecture of Pin1. Such modular architecture is common among cell-cycle proteins; thus, the WW–PPIase domain cross-talk mechanisms of Pin1 may be relevant for their mechanisms as well.

Activity and Affinity of Pin1 Variants

Pin1 is a peptidyl-prolyl isomerase responsible for isomerizing phosphorylated S/T-P motifs. Pin1 has two domains that each have a distinct ligand binding site, but only its PPIase domain has catalytic activity. Vast evidence supports interdomain allostery of Pin1, with binding of a ligand to its regulatory WW domain impacting activity in the PPIase domain. Many diverse studies have made mutations in Pin1 in order to elucidate interactions that are responsible for ligand binding, isomerase activity, and interdomain allostery. Here, we summarize these mutations and their impact on Pin1′s structure and function.

AtFKBP53: a chimeric histone chaperone with functional nucleoplasmin and PPIase domains

FKBP53 is one of the seven multi-domain FK506-binding proteins present in Arabidopsis thaliana, and it is known to get targeted to the nucleus. It has a conserved PPIase domain at the C-terminus and a highly charged N-terminal stretch, which has been reported to bind to histone H3 and perform the function of a histone chaperone. To better understand the molecular details of this PPIase with histone chaperoning activity, we have solved the crystal structures of its terminal domains and functionally characterized them. The C-terminal domain showed strong PPIase activity, no role in histone chaperoning and revealed a monomeric five-beta palm-like fold that wrapped over a helix, typical of an FK506-binding domain. The N-terminal domain had a pentameric nucleoplasmin-fold; making this the first report of a plant nucleoplasmin structure. Further characterization revealed the N-terminal nucleoplasmin domain to interact with H2A/H2B and H3/H4 histone oligomers, individually, as well as simultaneously, suggesting two different binding sites for H2A/H2B and H3/H4. The pentameric domain assists nucleosome assembly and forms a discrete complex with pre-formed nucleosomes; wherein two pentamers bind to a nucleosome.

Activity and Affinity of Pin1 Variants

Pin1 is a peptidyl-prolyl isomerase responsible for isomerizing phosphorylated S/T-P motifs. Pin1 has two domains that each have a distinct ligand binding site, but only its PPIase domain has catalytic activity. Vast evidence supports interdomain allostery of Pin1, with binding of a ligand to its regulatory WW domain impacting activity in the PPIase domain. Many diverse studies have made mutations in Pin1 in order to elucidate interactions that are responsible for ligand binding, isomerase activity, and interdomain allostery. Here, we summarize these mutations and their impact on Pin1’s structure and function.

Structural Analysis of the 42 kDa Parvulin of Trypanosoma brucei

Trypanosoma brucei is a unicellular eukaryotic parasite, which causes the African sleeping sickness in humans. The recently discovered trypanosomal protein Parvulin 42 (TbPar42) plays a key role in parasite cell proliferation. Homologues of this two-domain protein are exclusively found in protozoa species. TbPar42 exhibits an N-terminal forkhead associated (FHA)-domain and a peptidyl-prolyl-cis/trans-isomerase (PPIase) domain, both connected by a linker. Using NMR and X-ray analysis as well as activity assays, we report on the structures of the single domains of TbPar42, discuss their intra-molecular interplay, and give some initial hints as to potential cellular functions of the protein.

Noncovalent binding of a cyclic peptide inhibitor to the peptidyl-prolyl isomerase Pin1, explored by hydrogen exchange mass spectrometry

Pin1 is a peptidyl-prolyl isomerase (PPIase) that plays a central role in eukaryotic cell cycle regulation, making this protein an interesting target for cancer therapy. Pin1 exhibits high specificity for substrates where proline is preceded by phosphoserine or phosphothreonine. The protein comprises an N-terminal WW (tryptophan–tryptophan) domain and a C-terminal PPIase domain. The cyclic peptide [CRYPEVEIC] (square brackets are used to denote the cyclic structure) represents a lead compound for a new class of nonphosphorylated Pin1 inhibitors. Unfortunately, it has not been possible thus far to characterize the Pin1–[CRYPEVEIC] complex by X-ray crystallography. Thus, the exact binding mode remains unknown. The current work employs hydrogen/deuterium exchange mass spectrometry for gaining insights into the Pin1–[CRYPEVEIC] interactions. The WW domain shows extensive conformational dynamics, both in the presence and in the absence of ligand. In contrast, profound changes in deuteration kinetics are observed in the PPIase domain after the addition of [CRYPEVEIC]. The secondary structure elements β2, α3, and α4 exhibit markedly reduced deuteration, consistent with their postulated involvement in ligand binding. Unexpectedly, [CRYPEVEIC] destabilizes the range of residues 61–86, a segment that comprises basic side chains that normally interact with the substrate phosphate. This destabilization is likely caused by steric clashes with Y3 or E5 of the inhibitor. Ligand-induced destabilization has previously been reported for a few other proteins, but effects of this type are not very common. Our findings suggest that future crystallization trials on Pin1 variants deleted for residues in the 61–86 range might provide a path towards high-resolution X-ray structures of Pin1 bound to cyclic peptide inhibitors.

Structure and evolution of the spliceosomal peptidyl-prolylcis–transisomerase Cwc27

Cwc27 is a spliceosomal cyclophilin-type peptidyl-prolylcis–transisomerase (PPIase). Here, the crystal structure of a relatively protease-resistant N-terminal fragment of human Cwc27 containing the PPIase domain was determined at 2.0 Å resolution. The fragment exhibits a C-terminal appendix and resides in a reduced state compared with the previous oxidized structure of a similar fragment. By combining multiple sequence alignments spanning the eukaryotic tree of life and secondary-structure prediction, Cwc27 proteins across the entire eukaryotic kingdom were identified. This analysis revealed the specific loss of a crucial active-site residue in higher eukaryotic Cwc27 proteins, suggesting that the protein evolved from a prolyl isomerase to a pure proline binder. Noting a fungus-specific insertion in the PPIase domain, the 1.3 Å resolution crystal structure of the PPIase domain of Cwc27 fromChaetomium thermophilumwas also determined. Although structurally highly similar in the core domain, theC. thermophilumprotein displayed a higher thermal stability than its human counterpart, presumably owing to the combined effect of several amino-acid exchanges that reduce the number of long side chains with strained conformations and create new intramolecular interactions, in particular increased hydrogen-bond networks.