A Frustrated Binding Interface for Intrinsically Disordered Proteins

Intrinsically disordered proteins are very common in the eukaryotic proteome, and many of them are associated with diseases. Disordered proteins usually undergo a coupled binding and folding reaction and often interact with many different binding partners. Using double mutant cycles, we mapped the energy landscape of the binding interface for two interacting disordered domains and found it to be largely suboptimal in terms of interaction free energies, despite relatively high affinity. These data depict a frustrated energy landscape for interactions involving intrinsically disordered proteins, which is likely a result of their functional promiscuity.     

An Intrinsically Disordered Photosystem II Subunit,PsbO, Provides a Structural Template and a Sensor of the Hydrogen-bonding Network in Photosynthetic Water Oxidation

Photosystem II (PSII) is a membrane-bound enzyme that utilizes solar energy to catalyze the photooxidation of water. Molecular oxygen is evolved after four sequential light-driven oxidation reactions at the Mn₄CaO₅ oxygen-evolving complex, producing five sequentially oxidized states, S_n. PSII is composed of 17 membrane-spanning subunits and three extrinsic subunits, PsbP, PsbQ, and PsbO. PsbO is intrinsically disordered and plays a role in facilitation of the water oxidizing cycle. Native PsbO can be removed and substituted with recombinant PsbO, thereby restoring steady-state activity. In this report, we used reaction-induced Fourier transform infrared spectroscopy to obtain information concerning the role of PsbP, PsbQ, and PsbO during the S state cycle. Light-minus-dark difference spectra were acquired, monitoring structural changes associated with each accessible flash-induced S state transition in a highly purified plant PSII preparation (Triton X-100, octylthioglucoside). A comparison of S₂ minus S₁ spectra revealed that removal of PsbP and PsbQ had no significant effect on the data, whereas amide frequency and intensity changes were associated with PsbO removal. These data suggest that PsbO acts as an organizational template for the PSII reaction center. To identify any coupled conformational changes arising directly from PsbO, global ¹³C-PsbO isotope editing was employed. The reaction-induced Fourier transform infrared spectra of accessible S states provide evidence that PsbO spectral contributions are temperature (263 and 277 K) and S state dependent. These experiments show that PsbO undergoes catalytically relevant structural dynamics, which are coupled over long distance to hydrogen-bonding changes at the Mn₄CaO₅ cluster.     

Intrinsically Disordered Enamel Matrix Protein Ameloblastin Forms Ribbon-like Supramolecular Structures via an N-terminal Segment Encoded by Exon 5

Tooth enamel, the hardest tissue in the body, is formed by the evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins. The intrinsically disordered matrix protein ameloblastin (AMBN) is the most abundant nonamelogenin protein of the developing enamel and a key element for correct enamel formation. AMBN was suggested to be a cell adhesion molecule that regulates proliferation and differentiation of ameloblasts. Nevertheless, detailed structural and functional studies on AMBN have been substantially limited by the paucity of the purified nondegraded protein. With this study, we have developed a procedure for production of a highly purified form of recombinant human AMBN in quantities that allowed its structural characterization. Using size exclusion chromatography, analytical ultracentrifugation, transmission electron, and atomic force microscopy techniques, we show that AMBN self-associates into ribbon-like supramolecular structures with average widths and thicknesses of 18 and 0.34 nm, respectively. The AMBN ribbons exhibited lengths ranging from tens to hundreds of nm. Deletion analysis and NMR spectroscopy revealed that an N-terminal segment encoded by exon 5 comprises two short independently structured regions and plays a key role in self-assembly of AMBN.     

Multiple Interactions of the Intrinsically Disordered Region between the Helicase and Nuclease Domains of the Archaeal Hef Protein

Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein. We analyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and 10 candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.     

Expanding the Proteome of an RNA Virus by Phosphorylation of an Intrinsically Disordered Viral Protein

The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using ¹⁵N-, ¹³C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitis C virus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome.     

固有无序蛋白与蛋白质相互作用位点残基特征分析

本文对固有无序蛋白(IDPs)与其他蛋白质相互作用位点残基特征进行了研究.首先在数据库中选出满足条件的109条IDPs蛋白质链及与其他配体蛋白形成的299个IDPs-蛋白质复合物,然后提取复合物中作为相互作用位点的IDPs-蛋白质残基.这109条IDPs链中共含有50 031个氨基酸残基,其中处于作用位点的残基有4 822个.通过分析发现,20种氨基酸在形成IDPs-蛋白质相互作用位点残基时具有不同的倾向性,根据形成作用位点残基的倾向性,20种氨基酸可分成三大类:倾向型氨基酸(ILE、LEU、ARG、PHE、TYR、MET、TRP)、中间型氨基酸(GLN、GLU、THR、LYS、VAL、ASP、HIS)、非倾向型氨基酸(PRO、SER、GLY、ALA、ASN、CYS).研究结果还进一步表明,不同氨基酸在有序区域与无序区域形成IDPs-蛋白质作用位点残基的倾向性不同.其中,氨基酸TRP、LEU、ILE、CYS在有序和无序区域形成作用位点残基的差异性尤为明显,而氨基酸GLU、PHE、HIS、ALA则基本没有多大差别.对IDPs-蛋白质相互作用位点残基理化特征进行分析发现:疏水性强、侧链净电荷量较少、极性较小、溶剂可及性表面积较大、侧链体积较大、极化率较大的氨基酸比较倾向于形成作用位点残基.主成分分析结果显示,残基的极化率、侧链体积和溶剂可及表面积对作用位点残基影响最大.     

Structural dynamics and multiregion interactions in dynein-dynactin recognition

Cytoplasmic dynein is a 1.2-MDa multisubunit motor protein complex that, together with its activator dynactin, is responsible for the majority of minus end microtubule-based motility. Dynactin targets dynein to specific cellular locations, links dynein to cargo, and increases dynein processivity. These two macromolecular complexes are connected by a direct interaction between dynactin's largest subunit, p150(Glued), and dynein intermediate chain (IC) subunit. Here, we demonstrate using NMR spectroscopy and isothermal titration calorimetry that the binding footprint of p150(Glued) on IC involves two noncontiguous recognition regions, and both are required for full binding affinity. In apo-IC, the helical structure of region 1, the nascent helix of region 2, and the disorder in the rest of the chain are determined from coupling constants, amide-amide sequential NOEs, secondary chemical shifts, and various dynamics measurements. When bound to p150(Glued), different patterns of spectral exchange broadening suggest that region 1 forms a coiled-coil and region 2 a packed stable helix, with the intervening residues remaining disordered. In the 150-kDa complex of p150(Glued), IC, and two light chains, the noninterface segments remain disordered. The multiregion IC binding interface, the partial disorder of region 2 and its potential for post-translational modification, and the modulation of the length of the longer linker by alternative splicing may provide a basis for elegant and multifaceted regulation of binding between IC and p150(Glued). The long disordered linker between the p150(Glued) binding segments and the dynein light chain consensus sequences could also provide an attractive recognition platform for diverse cargoes.     

Intrinsic disorder in dynein intermediate chain modulates its interactions with NudE and dynactin

The functional diversity of cytoplasmic dynein is in part attributed to multiple interactions between noncatalytic dynein subunits and an array of regulatory proteins. This study focuses on the interaction between the dynein intermediate chain subunit (IC) and a dynein regulator protein (NudE). We use isothermal titration calorimetry and NMR spectroscopy to map their interacting sections to their respective N-terminal domains, which are predicted to form dimeric coiled-coils. Interestingly, the specific residues within IC that interact with NudE are a subset of the bi-segmental binding region reported for p150(Glued), a subunit of the dynein activator protein dynactin. Although the IC binding domains of both NudE and p150(Glued) form dimeric coiled-coils and bind IC at a common site, we observe distinct binding modes for each regulatory protein: 1) NudE binds region 1 of the bi-segmental binding footprint of p150(Glued), whereas p150(Glued) requires regions 1 and 2 to match the binding affinity of NudE with region 1 alone. 2) Compared with unbound IC, NudE-bound IC shows a slight increase in flexibility in region 2, in contrast to the increase in ordered structure observed for p150(Glued)-bound IC (Morgan, J. L., Song, Y., and Barbar, E. (2011) J. Biol. Chem. 286, 39349-39359). 3) Although NudE has a higher affinity for the common binding segment on IC, when all three proteins are in solution, IC preferentially binds p150(Glued). These results underscore the importance of a bi-segmental binding region of IC and disorder in region 2 and flanking linkers in selecting which regulatory protein binds IC.     

A Group 6 Late Embryogenesis Abundant Protein from Common Bean Is a Disordered Protein with Extended Helical Structure and Oligomer-forming Properties

Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association.     

Multiple Recognition Motifs in Nucleoporin Nup159 Provide a Stable and Rigid Nup159-Dyn2 Assembly

Dyn2 is the yeast ortholog of the molecular hub LC8, which binds disordered proteins and promotes their self-association and higher order assembly. Dyn2 is proposed to dimerize and stabilize the Nup82-Nsp1-Nup159 complex of the nuclear pore assembly through its interaction with nucleoporin Nup159. Nup159 has six LC8 recognition motifs separated by short linkers. NMR experiments reported here show that the Dyn2 binding domain of Nup159 is intrinsically disordered and that binding of one equivalent of Dyn2 dimer aligns two Nup159 chains along the full Dyn2 binding domain to form a bivalent scaffold that promotes binding of other Dyn2 dimers. Isothermal titration calorimetry of Dyn2 binding to Nup constructs of increasing lengths determine that the third LC8 recognition motifs does not bind Dyn2. A new approach to identifying active LC8 recognition motifs based on NMR-detected β-sheet propensities is presented. Isothermal titration calorimetry experiments also show that, due to unfavorable entropy changes, a Nup-Dyn2 complex with three Dyn2 dimers is more stable than the wild-type complex with five Dyn2 dimers. The calorimetric results argue that, from a thermodynamics perspective, only three Dyn2 dimers are needed for optimal stability and suggest that the evolutionary adaptation of multiple tandem LC8 recognition motifs imparts to the complex other properties such as rigidity and a kink in the rod-like structure. These findings extend the repertoire of functions of intrinsically disordered protein to fine-tuning and versatile assembly of higher order macromolecular complexes.     

Binding of Cysteine Synthase to the STAS Domain of Sulfate Transporter and Its Regulatory Consequences

The sulfate ion (SO₄²⁻) is transported into plant root cells by SO₄²⁻ transporters and then mostly reduced to sulfide (S²⁻). The S²⁻ is then bonded to O-acetylserine through the activity of cysteine synthase (O-acetylserine (thiol)lyase or OASTL) to form cysteine, the first organic molecule of the SO₄²⁻ assimilation pathway. Here, we show that a root plasma membrane SO₄²⁻ transporter of Arabidopsis, SULTR1;2, physically interacts with OASTL. The interaction was initially demonstrated using a yeast two-hybrid system and corroborated by both in vivo and in vitro binding assays. The domain of SULTR1;2 shown to be important for association with OASTL is called the STAS domain. This domain is at the C terminus of the transporter and extends from the plasma membrane into the cytoplasm. The functional relevance of the OASTL-STAS interaction was investigated using yeast mutant cells devoid of endogenous SO₄²⁻ uptake activity but co-expressing SULTR1;2 and OASTL. The analysis of SO₄²⁻ transport in these cells suggests that the binding of OASTL to the STAS domain in this heterologous system negatively impacts transporter activity. In contrast, the activity of purified OASTL measured in vitro was enhanced by co-incubation with the STAS domain of SULTR1;2 but not with the analogous domain of the SO₄²⁻ transporter isoform SULTR1;1, even though the SULTR1;1 STAS peptide also interacts with OASTL based on the yeast two-hybrid system and in vitro binding assays. These observations suggest a regulatory model in which interactions between SULTR1;2 and OASTL coordinate internalization of SO₄²⁻ with the energetic/metabolic state of plant root cells.     

Characterization of the interactions between the nucleoprotein and the phosphoprotein of Henipavirus

The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (N(TAIL)) are both intrinsically disordered. Here we show that Henipavirus N(TAIL) domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (P(XD)) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that N(TAIL) and P(XD) form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M and has a K(D) in the μM range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that P(XD) triggers an increase in the α-helical content of N(TAIL). Using fluorescence spectroscopy, we show that P(XD) has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus N(TAIL) domain, thus arguing for the lack of stable contacts between the C termini of N(TAIL) and P(XD). Finally, we present a tentative structural model of the N(TAIL)-P(XD) interaction in which a short, order-prone region of N(TAIL) (α-MoRE; amino acids 473-493) adopts an α-helical conformation and is embedded between helices α2 and α3 of P(XD), leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the N(TAIL)-P(XD) interaction as a valuable target for rational antiviral approaches.     

Neurogranin Alters the Structure and Calcium Binding Properties of Calmodulin

Neurogranin (Ng) is a member of the IQ motif class of calmodulin (CaM)-binding proteins, and interactions with CaM are its only known biological function. In this report we demonstrate that the binding affinity of Ng for CaM is weakened by Ca²⁺ but to a lesser extent (2–3-fold) than that previously suggested from qualitative observations. We also show that Ng induced a >10-fold decrease in the affinity of Ca²⁺ binding to the C-terminal domain of CaM with an associated increase in the Ca²⁺ dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca²⁺ binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca²⁺-saturated. Domain mapping using synthetic peptides indicated that the IQ motif of Ng is a poor mimetic of the intact protein and that the acidic sequence just N-terminal to the IQ motif plays an important role in reproducing Ng-mediated decreases in the Ca²⁺ binding affinity of CaM. Using NMR, full-length Ng was shown to make contacts largely with residues in the C-domain of CaM, although contacts were also detected in residues in the N-terminal domain. Together, our results can be consolidated into a model where Ng contacts residues in the N- and C-lobes of both apo- and Ca²⁺-bound CaM and that although Ca²⁺ binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca²⁺ binding to the C-terminal lobe of CaM.     

Involvement of the N-terminal B-box Domain of Arabidopsis BBX32 Protein in Interaction with Soybean BBX62 Protein

Previous studies have demonstrated that Arabidopsis thaliana BBX32 (AtBBX32) represses light signaling in A. thaliana and that expression of AtBBX32 in soybean increases grain yield in multiple locations and multiyear field trials. The BBX32 protein is a member of the B-box zinc finger family from A. thaliana and contains a single conserved Zn(2+)-binding B-box domain at the N terminus. Although the B-box domain is predicted to be involved in protein-protein interactions, the mechanism of interaction is poorly understood. Here, we provide in vitro and in vivo evidence demonstrating the physical and functional interactions of AtBBX32 with another B-box protein, soybean BBX62 (GmBBX62). Deletion analysis and characterization of the purified B-box domain indicate that the N-terminal B-box region of AtBBX32 interacts with GmBBX62. Computational modeling and site-directed mutagenesis of the AtBBX32 B-box region identified specific residues as critical for mediating the interaction between AtBBX32 and GmBBX62. This study defines the plant B-box as a protein interaction domain and offers novel insight into its role in mediating specific protein-protein interactions between different plant B-box proteins.     

Identification and Analysis of a Novel Dimerization Domain Shared by Various Members of c-Jun N-terminal Kinase (JNK) Scaffold Proteins

Mitogen-activated protein kinases (MAPKs) form a kinase tier module in which MAPK, MAP2K, and MAP3K are held by scaffold proteins. The scaffold proteins serve as a protein platform for selective and spatial kinase activation. The precise mechanism by which the scaffold proteins function has not yet been fully explained. WDR62 is a novel scaffold protein of the c-Jun N-terminal kinase (JNK) pathway. Recessive mutations within WDR62 result in severe cerebral cortical malformations. One of the WDR62 mutant proteins found in a patient with microcephaly encodes a C-terminal truncated protein that fails to associate efficiently with JNK and MKK7β1. The present article shows that the WDR62 C-terminal region harbors a novel dimerization domain composed of a putative loop-helix domain that is necessary and sufficient for WDR62 dimerization and is critical for its scaffolding function. The loop-helix domain is highly conserved between orthologues and is also shared by the JNK scaffold protein, JNKBP1/MAPKBP1. Based on the high sequence conservation of the loop-helix domain, our article shows that MAPKBP1 homodimerizes and heterodimerizes with WDR62. Endogenous WDR62 and MAPKBP1 co-localize to stress granules following arsenite treatment, but not during mitosis. This study proposes another layer of complexity, in which coordinated activation of signaling pathways is mediated by the association between the different JNK scaffold proteins depending on their biological function.     

The Tat System for Membrane Translocation of Folded Proteins Recruits the Membrane-stabilizing Psp Machinery in Escherichia coli

Tat systems transport folded proteins across energized membranes of bacteria, archaea, and plant plastids. In Escherichia coli, TatBC complexes recognize the transported proteins, and TatA complexes are recruited to facilitate transport. We achieved an abstraction of TatA from membranes without use of detergents and observed a co-purification of PspA, a membrane-stress response protein. The N-terminal transmembrane domain of TatA was required for the interaction. Electron microscopy displayed TatA complexes in direct contact with PspA. PspB and PspC were important for the TatA-PspA contact. The activator protein PspF was not involved in the PspA-TatA interaction, demonstrating that basal levels of PspA already interact with TatA. Elevated TatA levels caused membrane stress that induced a strictly PspBC- and PspF-dependent up-regulation of PspA. TatA complexes were found to destabilize membranes under these conditions. At native TatA levels, PspA deficiency clearly affected anaerobic TMAO respiratory growth, suggesting that energetic costs for transport of large Tat substrates such as TMAO reductase can become growth limiting in the absence of PspA. The physiological role of PspA recruitment to TatA may therefore be the control of membrane stress at active translocons.     

Membrane anchoring and interaction between transmembrane domains are crucial for K+ channel function

The small viral channel Kcv is a Kir-like K(+) channel of only 94 amino acids. With this simple structure, the tetramer of Kcv represents the pore module of all complex K(+) channels. To examine the structural contribution of the transmembrane domains (TMDs) to channel function, we performed Ala scanning mutagenesis of the two domains and tested the functionality of the mutants in a yeast complementation assay. The data reveal, in combination with computational models, that the upper halves of both TMDs, which face toward the external medium, are rather rigid, whereas the inner parts are more flexible. The rigidity of the outer TMD is conferred by a number of essential aromatic amino acids that face the membrane and probably anchor this domain in the bilayer. The inner TMD is intimately connected with the rigid part of the outer TMD via π···π interactions between a pair of aromatic amino acids. This structural principle is conserved within the viral K(+) channels and also present in Kir2.2, implying a general importance of this architecture for K(+) channel function.