Restricted domain mobility in the Candida albicans Ess1 prolyl isomerase

Ess1 is a peptidyl prolyl cis/trans isomerase that is required for virulence of the pathogenic fungi Candida albicans and Cryptococcus neoformans. The enzyme isomerizes the phospho-Ser-Pro linkages in the C-terminal domain of RNA polymerase II. Its human homolog, Pin1, has been implicated in a wide range of human diseases, including cancer and Alzheimer's disease. Crystallographic and NMR studies have demonstrated that the sequence linking the catalytic isomerase domain and the substrate binding WW domain of Pin1 is unstructured and that the two domains are only loosely associated in the absence of the substrate. In contrast, the crystal structure of C. albicans Ess1 revealed a highly ordered linker that contains a three turn α-helix and extensive association between the two tightly juxtaposed domains. In part to address the concern that the marked differences in the domain interactions for the human and fungal structures might reflect crystal lattice effects, NMR chemical shift analysis and ¹⁵N relaxation measurements have been employed to confirm that the linker of the fungal protein is highly ordered in solution. With the exception of two loops within the active site of the isomerase domain, the local backbone geometry observed in the crystal structure appears to be well preserved throughout the protein chain. The marked differences in interdomain interactions and linker flexibility between the human and fungal enzymes provide a structural basis for therapeutic targeting of the fungal enzymes.     

NMR relaxation unravels interdomain crosstalk of the two domain prolyl isomerase and chaperone SlyD

The dynamics of the two domain prolyl-peptidyl cis/trans isomerase and chaperone SlyD was studied on a ps-to-ns time scale to correlate dynamic changes with the catalytic function. (15)N transversal and longitudinal relaxation rates as well as heteronuclear Overhauser effects were determined at different temperatures for Escherichia coli SlyD (EcSlyD) and for Thermus thermophilus SlyD (TtSlyD). With the well established extended Lipari-Szabo approach, the order parameter, S(2), the internal correlation time, τ(e), the exchange rate, R(ex), of the backbone amide protons, and the overall molecular tumbling time, τ(m), were determined. The study was extended to a relaxation analysis of the peptide bound state for both SlyD species. We found highly different relaxation and dynamic behavior of the two domains for free SlyD. Surprisingly, in the presence of a substrate for the chaperone domain, the ps-to-ns dynamics in the remote center of the prolyl-peptidyl cis/trans isomerization domain increases. We observed this crosstalk between the two domains for both EcSlyD and TtSlyD.     

Three binding sites in protein-disulfide isomerase cooperate in collagen prolyl 4-hydroxylase tetramer assembly

Protein-disulfide isomerase (PDI) is a modular polypeptide consisting of four domains, a, b, b', and a'. It is a ubiquitous protein folding catalyst that in addition functions as the beta-subunit in vertebrate collagen prolyl 4-hydroxylase (C-P4H) alpha(2)beta(2) tetramers. We report here that point mutations in the primary peptide substrate binding site in the b' domain of PDI did not inhibit C-P4H assembly. Based on sequence conservation, additional putative binding sites were identified in the a and a' domains. Mutations in these sites significantly reduced C-P4H tetramer assembly, with the a domain mutations generally having the greater effect. When the a or a' domain mutations were combined with the b' domain mutation I272W tetramer assembly was further reduced, and more than 95% of the assembly was abolished when mutations in the three domains were combined. The data indicate that binding sites in three PDI domains, a, b', and a', contribute to efficient C-P4H tetramer assembly. The relative contributions of these sites were found to differ between Caenorhabditis elegans C-P4H alphabeta dimer and human alpha(2)beta(2) tetramer formation.     

Thermodynamics of phosphopeptide binding to the human peptidyl prolyl cis/trans isomerase Pin1

Proteins containing phosphorylated Ser/Thr-Pro motifs play key roles in numerous regulatory processes in the cell. The peptidyl prolyl cis/trans isomerase Pin1 specifically catalyzes the conformational transition of phosphorylated Ser/Thr-Pro motifs. Here we report the direct analysis of the thermodynamic properties of the interaction of the PPIase Pin1 with its substrate-analogue inhibitor Ac-Phe-D-Thr(PO3H2)-Pip-Nal-Gln-NH2 specifically targeted to the PPIase active site based on the combination of isothermal titration calorimetry and studies on inhibition of enzymatic activity of wt Pin1 and active site variants. Determination of the thermodynamic parameters revealed an enthalpically and entropically favored interaction characterized by binding enthalpy deltaH(ITC) of -6.3 +/- 0.1 kcal mol(-1) and a TdeltaS(ITC) of 4.1 +/- 0.1 kcal mol(-1). The resulting dissociation constant KD for binding of the peptidic inhibitor with 1.8 x 10(-8) M resembles the dissociation constant of a Pin1 substrate in the transition state, suggesting a transition state analogue conformation of the bound inhibitor. The strongly decreased affinity of Pin1 for ligand at increasing ionic strength implicates that the potential of bidentate binding of a substrate protein by the PPIase and the WW domain of Pin1 may be required to deploy improved efficiency and specificity of Pin1 under conditions of physiological ionic strength.     

The role of backbone motions in ligand binding to the c-Src SH3 domain.

The Src homology 3 (SH3) domain of pp60(c-src) (Src) plays dual roles in signal transduction, through stabilizing the repressed form of the Src kinase and through mediating the formation of activated signaling complexes. Transition of the Src SH3 domain between a variety of binding partners during progression through the cell cycle requires adjustment of a delicate free energy balance. Although numerous structural and functional studies of SH3 have provided an in-depth understanding of structural determinants for binding, the origins of binding energy in SH3-ligand interactions are not fully understood. Considering only the protein-ligand interface, the observed favorable change in standard enthalpy (DeltaH=-9.1 kcal/mol) and unfavorable change in standard entropy (TDeltaS=-2.7 kcal/mol) upon binding the proline-rich ligand RLP2 (RALPPLPRY) are inconsistent with the predominantly hydrophobic interaction surface. To investigate possible origins of ligand binding energy, backbone dynamics of free and RLP2-bound SH3 were performed via (15)N NMR relaxation and hydrogen-deuterium (H/(2)H) exchange measurements. On the ps-ns time scale, assuming uncorrelated motions, ligand binding results in a significant reduction in backbone entropy (-1.5(+/-0.6) kcal/mol). Binding also suppresses motions on the micros-ms time scale, which may additionally contribute to an unfavorable change in entropy. A large increase in protection from H/(2)H exchange is observed upon ligand binding, providing evidence for entropy loss due to motions on longer time scales, and supporting the notion that stabilization of pre-existing conformations within a native state ensemble is a fundamental paradigm for ligand binding. Observed changes in motion on all three time scales occur at locations both near and remote from the protein-ligand interface. The propagation of ligand binding interactions across the SH3 domain has potential consequences in target selection through altering both free energy and geometry in intact Src, and suggests that looking beyond the protein-ligand interface is essential in understanding ligand binding energetics.     

CELF proteins regulate CFTR pre-mRNA splicing: essential role of the divergent domain of ETR-3

Cystic fibrosis is a prominent genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Among the many disease-causing alterations are pre-mRNA splicing defects that can hamper mandatory exon inclusion. CFTR exon 9 splicing depends in part on a polymorphic UG(m)U(n) sequence at the end of intron 8, which can be bound by TDP-43, leading to partial exon 9 skipping. CELF proteins, like CUG-BP1 and ETR-3, can also bind UG repeats and regulate splicing. We show here that ETR-3, but not CUG-BP1, strongly stimulates exon 9 skipping, although both proteins bind efficiently to the same RNA motif as TDP-43 and with higher affinity. We further show that the skipping of this exon may be due to the functional antagonism between U2AF⁶⁵ and ETR-3 binding onto the polymorphic U or UG stretch, respectively. Importantly, we demonstrate that the divergent domain of ETR-3 is critical for CFTR exon 9 skipping, as shown by deletion and domain-swapping experiments. We propose a model whereby several RNA-binding events account for the complex regulation of CFTR exon 9 inclusion, with strikingly distinct activities of ETR-3 and CUG-BP1, related to the structure of their divergent domain.     

Folding and association of the antibody domain CH3: prolyl isomerization preceeds dimerization.

The simplest naturally occurring model system for studying immunoglobulin folding and assembly is the non-covalent homodimer formed by the C-terminal domains (CH3) of the heavy chains of IgG. Here, we describe the structure of recombinant CH3 dimer as determined by X-ray crystallography and an analysis of the folding pathway of this protein.Under conditions where prolyl isomerization does not contribute to the folding kinetics, formation of the beta-sandwich structure is the rate-limiting step. beta-Sheet formation of CH3 is a slow process, even compared to other antibody domains, while the subsequent association of the folded monomers is fast. After long-time denaturation, the majority of the unfolded CH3 molecules reaches the native state in two serial reactions, involving the re-isomerization of the Pro35-peptide bond to the cis configuration. The species with the wrong isomer accumulate as a monomeric intermediate. Importantly, the isomerization to the correct cis configuration is the prerequisite for dimerization of the CH3 domain. In contrast, in the Fab fragment of the same antibody, prolyl isomerization occurs after dimerization demonstrating that within one protein, comprised of highly homologous domains, both the kinetics of beta-sandwich formation and the stage at which prolyl isomerization occurs during the folding process can be completely different.     

The KH domain of the branchpoint sequence binding protein determines specificity for the pre-mRNA branchpoint sequence.

The yeast and mammalian branchpoint sequence binding proteins (BBP and mBBP/SF1) contain both KH domain and Zn knuckle RNA-binding motifs. The single KH domain of these proteins is sufficient for specific recognition of the pre-mRNA branchpoint sequence (BPS). However, an interaction is only apparent if one or more accessory modules are present to increase binding affinity. The Zn knuckles of BBP/mBBP can be replaced by an RNA-binding peptide derived from the HIV-1 nucleocapsid protein or by an arginine-serine (RS)7 peptide, without loss of specificity. Only the seven-nucleotide branchpoint sequence and two nucleotides to either side are necessary for RNA binding to the chimeric proteins. Therefore, we propose that all three of these accessory RNA-binding modules bind the phosphate backbone, whereas the KH domain interacts specifically with the bases of the BPS. Proteins and protein complexes with multiple RNA-binding motifs are frequent, suggesting that an intimate collaboration between two or more motifs will be a general theme in RNA-protein interactions.     

The prolyl isomerase domain of PpiD from Escherichia coli shows a parvulin fold but is devoid of catalytic activity

PpiD is a periplasmic folding helper protein of Escherichia coli. It consists of an N‐terminal helix that anchors PpiD in the inner membrane near the SecYEG translocon, followed by three periplasmic domains. The second domain (residues 264–357) shows homology to parvulin‐like prolyl isomerases. This domain is a well folded, stable protein and follows a simple two‐state folding mechanism. In its solution structure, as determined by NMR spectroscopy, it resembles most closely the first parvulin domain of the SurA protein, which resides in the periplasm of E. coli as well. A previously reported prolyl isomerase activity of PpiD could not be reproduced when using improved protease‐free peptide assays or assays with refolding proteins as substrates. The parvulin domain of PpiD interacts, however, with a proline‐containing tetrapeptide, and the binding site, as identified by NMR resonance shift analysis, colocalized with the catalytic sites of other parvulins. In its structure, the parvulin domain of PpiD resembles most closely the inactive first parvulin domain of SurA, which is part of the chaperone unit of this protein and presumably involved in substrate recognition.     

Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase     

The role of subtype-specific ligand binding and the C-tail domain in dimer formation of human somatostatin receptors

G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors. Several GPCRs have been documented to dimerize with resulting changes in pharmacology. We have previously reported by means of photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence correlation spectroscopic (FCS) analysis in live cells, that human somatostatin receptor (hSSTR) 5 could both homodimerize and heterodimerize with hSSTR1 in the presence of the agonist SST-14. In contrast, hSSTR1 remained monomeric when expressed alone regardless of agonist exposure in live cells. In an effort to elucidate the role of ligand and receptor subtypes in heterodimerization, we have employed both pb-FRET microscopy and Western blot on cells stably co-expressing hSSTR1 and hSSTR5 treated with subtype-specific agonists. Here we provide evidence that activation of hSSTR5 but not hSSTR1 is necessary for heterodimeric assembly. This property was also reflected in signaling as shown by increases in adenylyl cyclase coupling efficiencies. Furthermore, receptor C-tail chimeras allowed for the identification of the C-tail as a determinant for dimerization. Finally, we demonstrate that heterodimerization is subtype-selective involving ligand-induced conformational changes in hSSTR5 but not hSSTR1 and could be attributed to molecular events occurring at the C-tail. Understanding the mechanisms by which GPCRs dimerize holds promise for improvements in drug design and efficacy.     

Human interleukin-3 contains a discontinuous zinc binding domain.

The primary structure of human interleukin-3 contains two amino acid consensus sequences at Glutamate 22- Histidine 26 and Histidine 95-Histidine 98, that are characteristic for zinc binding proteins. Therefore, the hypothesis was tested that human interleukin-3 binds zinc specifically by either one or both sequences. Protein dotblotting, followed by probing with radioactive zinc demonstrated specific zinc binding of interleukin-3. Metal specificity was confirmed by competition experiments with 12 other divalent- and trivalent metal ions. Protease treatment combined with plasma desorption mass spectrometry was used to localize the zinc binding domain. Specific zinc binding was restricted to a fragment composed of Threonine 11-Lysine 28 and Asparagine 80-Lysine 100. It was found to decrease by a factor of five when either of these two amino acid stretches was missing. It is concluded that human interleukin-3 is a zinc binding protein. Interleukin-3 zinc binding capacity is largely determined by both moieties of the protein that contain the consensus sequences. In addition we propose that the zinc binding of hIL-3 is involved in (de)phosphorylation of the hIL-3 receptor.