Kenji Sugase

Mechanism of coupled folding and binding of an intrinsically disordered protein

Protein folding and binding are analogous processes, in which the protein ‘searches’ for favourable intramolecular or intermolecular interactions on a funnelled energy landscape. Many eukaryotic proteins are disordered under physiological conditions, and fold into ordered structures only on binding to their cellular targets. The mechanism by which folding is coupled to binding is poorly understood, but it has been hypothesized on theoretical grounds that the binding kinetics may be enhanced by a ‘fly-casting’ effect, where the disordered protein binds weakly and non-specifically to its target and folds as it approaches the cognate binding site. Here we show, using NMR titrations and 15N relaxation dispersion, that the phosphorylated kinase inducible activation domain (pKID) of the transcription factor CREB forms an ensemble of transient encounter complexes on binding to the KIX domain of the CREB binding protein. The encounter complexes are stabilized primarily by non-specific hydrophobic contacts, and evolve by way of an intermediate to the fully bound state without dissociation from KIX. The carboxy-terminal helix of pKID is only partially folded in the intermediate, and becomes stabilized by intermolecular interactions formed in the final bound state. Future applications of our method will provide new understanding of the molecular mechanisms by which intrinsically disordered proteins perform their diverse biological functions.

Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding

Significance Intrinsically disordered proteins play a central role in cellular signaling and regulatory networks. Many regulatory proteins contain disordered recognition motifs that fold upon binding to their cellular targets, by mechanisms that are poorly understood. Here we show that the disordered transactivation domain of the transcription factor c-Myb binds to the KIX domain of the transcriptional coactivator cAMP-response element binding (CREB-binding) protein (CBP) by a mechanism that involves elements of conformational selection and induced fit. In contrast, the phosphorylated kinase-inducible activation domain of CREB binds to the same site on KIX by an induced-fit mechanism. The intrinsic secondary structure propensities of these two disordered proteins determine their binding mechanisms, consistent with their functions as inducible and constitutive transcriptional activators. Intrinsically disordered proteins (IDPs) frequently function in protein interaction networks that regulate crucial cellular signaling pathways. Many IDPs undergo transitions from disordered conformational ensembles to folded structures upon binding to their cellular targets. Several possible binding mechanisms for coupled folding and binding have been identified: folding of the IDP after association with the target (“induced fit”), or binding of a prefolded state in the conformational ensemble of the IDP to the target protein (“conformational selection”), or some combination of these two extremes. The interaction of the intrinsically disordered phosphorylated kinase-inducible domain (pKID) of the cAMP-response element binding (CREB) protein with the KIX domain of a general transcriptional coactivator CREB-binding protein (CBP) provides an example of the induced-fit mechanism. Here we show by NMR relaxation dispersion experiments that a different intrinsically disordered ligand, the transactivation domain of the transcription factor c-Myb, interacts with KIX at the same site as pKID but via a different binding mechanism that involves elements of conformational selection and induced fit. In contrast to pKID, the c-Myb activation domain has a strong propensity for spontaneous helix formation in its N-terminal region, which binds to KIX in a predominantly folded conformation. The C-terminal region of c-Myb exhibits a much smaller helical propensity and likely folds via an induced-fit process after binding to KIX. We propose that the intrinsic secondary structure propensities of pKID and c-Myb determine their binding mechanisms, consistent with their functions as inducible and constitutive transcriptional activators.

Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance

Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95u2009Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem–haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer.

Fast and accurate fitting of relaxation dispersion data using the flexible software package GLOVE

Relaxation dispersion spectroscopy is one of the most widely used techniques for the analysis of protein dynamics. To obtain a detailed understanding of the protein function from the view point of dynamics, it is essential to fit relaxation dispersion data accurately. The grid search method is commonly used for relaxation dispersion curve fits, but it does not always find the global minimum that provides the best-fit parameter set. Also, the fitting quality does not always improve with increase of the grid size although the computational time becomes longer. This is because relaxation dispersion curve fitting suffers from a local minimum problem, which is a general problem in non-linear least squares curve fitting. Therefore, in order to fit relaxation dispersion data rapidly and accurately, we developed a new fitting program called GLOVE that minimizes global and local parameters alternately, and incorporates a Monte-Carlo minimization method that enables fitting parameters to pass through local minima with low computational cost. GLOVE also implements a random search method, which sets up initial parameter values randomly within user-defined ranges. We demonstrate here that the combined use of the three methods can find the global minimum more rapidly and more accurately than grid search alone.

Structural element responsible for the Fe(III)-phytosiderophore specific transport by HvYS1 transporter in barley.

Hordeum vulgare L. yellow stripe 1 (HvYS1) is a selective transporter for Fe(III)–phytosiderophores, involved in primary iron acquisition from soils in barley roots. In contrast, Zea mays yellow stripe 1 (ZmYS1) in maize possesses broad substrate specificity, despite a high homology with HvYS1. Here we revealed, by assessing the transport activity of a series of HvYS1–ZmYS1 chimeras, that the outer membrane loop between the sixth and seventh transmembrane regions is essential for substrate specificity. Circular dichroism spectra indicated that a synthetic peptide corresponding to the loop of HvYS1 forms an α‐helix in solution, whereas that of ZmYS1 is flexible. We propose that the structural difference at this particular loop determines the substrate specificity of the HvYS1 transporter.

Calcitonin in a protochordate, Ciona intestinalis – the prototype of the vertebrate calcitonin/calcitonin gene-related peptide superfamily

The calcitonin (CT)/CT gene‐related peptides (CGRPs) constitute a large peptide family in vertebrates. However, no CT/CGRP superfamily members have so far been identified in invertebrates, and the evolutionary process leading to the diverse vertebrate CT/CGRP superfamily members remains unclear. In this study, we have identified an authentic invertebrate CT, Ci‐CT, in the ascidian Cionau2003intestinalis, which is the phylogenetically closest invertebrate chordate to vertebrates. The amino acid sequence of Ci‐CT was shown to display high similarity to those of vertebrate CTs and to share CT consensus motifs, including the N‐terminal circular region and C‐terminal amidated proline. Furthermore, the Ci‐CT gene was found to be the only Ciona CT/CGRP superfamily gene. Ci‐CT also exhibited less potent, but significant, activation of the human CT receptor, as compared with salmon CT. Physiological analysis revealed that Ci‐CT reduced the osteoclastic activity that is specific to vertebrate CTs. CD analysis demonstrated that Ci‐CT weakly forms an α‐helix structure. These results provide evidence that the CT/CGRP superfamily is essentially conserved in ascidians as well as in vertebrates, and indicate that Ci‐CT is a prototype of vertebrate CT/CGRP superfamily members. Moreover, expression analysis demonstrated that Ci‐CT is expressed in more organs than vertebrate CTs in the cognate organs, suggesting that an original CT/CGRP superfamily member gene was also expressed in multiple organs, and each CT/CGRP superfamily member acquired its current specific tissue distribution and physiological role concomitantly with diversification of the CT/CGRP superfamily during the evolution of chordates. This is the first report on a CT/CGRP superfamily member in invertebrates.

Solution Structure of the Ubiquitin-associated (UBA) Domain of Human Autophagy Receptor NBR1 and Its Interaction with Ubiquitin and Polyubiquitin

Background: The autophagic receptor NBR1 is commonly found in ubiquitin-positive inclusions in neurodegenerative diseases. Results: Molecular recognition of ubiquitin and polyubiquitin by NBR1 is described. Conclusion: The ubiquitin-associated domain of NBR1 shows unexpectedly high affinity for monoubiquitin but lacks polyubiquitin linkage specificity. Significance: NBR1 may be highly efficient at forming intracellular inclusions with ubiquitylated proteins via non-linkage-specific association with ubiquitin. NBR1 (neighbor of BRCA1 gene 1) is a protein commonly found in ubiquitin-positive inclusions in neurodegenerative diseases. Due to its high architectural similarity to the well studied autophagy receptor protein p62/SQSTM1, NBR1 has been thought to analogously bind to ubiquitin-marked autophagic substrates via its C-terminal ubiquitin-associated (UBA) domain and deliver them to autophagosomes for degradation. Unexpectedly, we find that NBR1 differs from p62 in its UBA structure and accordingly in its interaction with ubiquitin. Structural differences are observed on helix α-3, which is tilted farther from helix α-2 and extended by approximately one turn in NBR1. This results not only in inhibition of a p62-type self-dimerization of NBR1 UBA but also in a significantly higher affinity for monoubiquitin as compared with p62 UBA. Importantly, the NBR1 UBA-ubiquitin complex structure shows that the negative charge of the side chain in front of the conserved MGF motif in the UBA plays an integral role in the recognition of ubiquitin. In addition, NMR and isothermal titration calorimetry experiments show that NBR1 UBA binds to each monomeric unit of polyubiquitin with similar affinity and by the same surface used for binding to monoubiquitin. This indicates that NBR1 lacks polyubiquitin linkage-type specificity, in good agreement with the nonspecific linkages observed in intracellular ubiquitin-positive inclusions. Consequently, our results demonstrate that the structural differences between NBR1 UBA and p62 UBA result in a much higher affinity of NBR1 for ubiquitin, which in turn suggests that NBR1 may form intracellular inclusions with ubiquitylated autophagic substrates more efficiently than p62.

Quantitative analysis of protein–ligand interactions by NMR

Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used to analyze population-averaged NMR quantities. Essentially, to apply NMR successfully, both the type of experiment and equation to fit the data must be carefully and specifically chosen for the protein-ligand interaction under analysis. In this review, we first explain the exchange regimes and kinetic models of protein-ligand interactions, and then describe the NMR methods that quantitatively analyze these specific interactions.

The monomer-seed interaction mechanism in the formation of the β2-microglobulin amyloid fibril clarified by solution NMR techniques.

Amyloid fibrils are proteinous aggregates associated with various diseases, including Alzheimers disease, type II diabetes, and dialysis-related amyloidosis. It is generally thought that, during the progression of these diseases, a precursor peptide or protein assumes a partially denatured structure, which interacts with the fibril seed to change into the final amyloid form. β2-Microglobulin (β2m), associated with dialysis-related amyloidosis, is known to form amyloid fibrils at low pH via a partially structured state. However, the molecular mechanism by which the conformation of β2m changes from the precursor to the final fibril structure is poorly understood. We performed various NMR experiments to characterize acid-denatured β2m. The analysis of the transverse relaxation rates revealed that acid-denatured β2m undergoes a structural exchange with an extensively unfolded form. The results of transferred cross-saturation experiments indicated that residues with a residual structure in the acid-denatured state are associated with the interaction with the fibril seed. Our experimental data suggest the partially structured state to be activated to become extensively unfolded, in which state the hydrophobic residues are exposed and associate with the seed. Our results provide general information about the extension of amyloid fibrils.

Dynamic changes in CCAN organization through CENP-C during cell-cycle progression

Dynamic changes in CCAN organization during progression of the cell cycle are examined in chicken DT40 cells. CENP-C166-324 is sufficient for interphase centromere localization through association with CENP-L-N, and CENP-C643-864 is essential for mitotic centromere localization through binding to CENP-A nucleosomes.