Functional dissection of an intrinsically disordered protein: Understanding the roles of different domains of Knr4 protein in protein�Cprotein interactions

Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N‐terminal and large natively unfolded C‐terminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4. Here, we used an in vivo two‐hybrid system approach and an in vitro surface plasmon resonance (BIAcore) technique to compare the interaction level of different Knr4 deletion variants with given protein partners. We demonstrate the indispensability of the N‐terminal domain of Knr4 for the interactions. On the other hand, presence of the unstructured C‐terminal domain has a negative effect on the interaction strength. In protein interactions networks, the most highly connected proteins or “hubs” are significantly enriched in unstructured regions, and among them the transient hub proteins contain the largest and most highly flexible regions. The results presented here of our analysis of Knr4 protein suggest that these large disordered regions are not always involved in promoting the protein–protein interactions of hub proteins, but in some cases, might rather inhibit them. We propose that this type of regions could prevent unspecific protein interactions, or ensure the correct timing of occurrence of transient interactions, which may be of crucial importance for different signaling and regulation processes.     

Binding mode analysis of a major T3SS translocator protein PopB with its chaperone PcrH from Pseudomonas aeruginosa

Pseudomonas aeruginosa, a Gram‐negative pathogen uses a specialized set of Type III secretion system (T3SS) translocator proteins to establish virulence in the host cell. An understanding of the factors that govern translocation by the translocator protein–chaperone complex is thus of immense importance. In this work, experimental and computational techniques were used to probe into the structure of the major translocator protein PopB from P. aeruginosa and to identify the important regions involved in functioning of the translocator protein. This study reveals that the binding sites of the common chaperone PcrH, needed for maintenance of the translocator PopB within the bacterial cytoplasm, which are primarily localized within the N‐terminal domain. However, disordered and flexible residues located both at the N‐ and C‐terminal domains are also observed to be involved in association with the chaperone. This intrinsic disorderliness of the terminal domains is conserved for all the major T3SS translocator proteins and is functionally important to maintain the intrinsically disordered state of the translocators. Our experimental and computational analyses suggest that a “disorder‐to‐order” transition of PopB protein might take place upon PcrH binding. The long helical coiled‐coil part of PopB protein perhaps helps in pore formation while the flexible apical region is involved in chaperone interaction. Thus, our computational model of translocator protein PopB and its binding analyses provide crucial functional insights into the T3SS translocation mechanism. Proteins 2014; 82:3273–3285. © 2014 Wiley Periodicals, Inc.     

The intermembrane space domain of Tim23 is intrinsically disordered with a distinct binding region for presequences

Proteins targeted to the mitochondrial matrix are translocated through the outer and the inner mitochondrial membranes by two protein complexes, the translocase of the outer membrane (TOM) and one of the translocases of the inner membrane (TIM23). The protein Tim23, the core component of TIM23, consists of an N‐terminal, soluble domain in the intermembrane space (IMS) and a C‐terminal domain that forms the import pore across the inner membrane. Before translocation proceeds, precursor proteins are recognized by the N‐terminal domain of Tim23, Tim23N (residues 1–96). By using NMR spectroscopy, we show that Tim23N is a monomeric protein belonging to the family of intrinsically disordered proteins. Titrations of Tim23N with two presequences revealed a distinct binding region of Tim23N formed by residues 71–84. In a charge‐hydropathy plot containing all soluble domains of TOM and TIM23, Tim23N was found to be the only domain with more than 40 residues in the IMS that is predicted to be intrinsically disordered, suggesting that Tim23N might function as hub in the mitochondrial import machinery protein network.     

A flexible C‐terminal linker is required for proper FtsZ assembly in vitro and cytokinetic ring formation in vivo

Assembly of the cytoskeletal protein FtsZ into a ring‐like structure is required for bacterial cell division. Structurally, FtsZ consists of four domains: the globular N‐terminal core, a flexible linker, 8–9 conserved residues implicated in interactions with modulatory proteins, and a highly variable set of 4–10 residues at its very C terminus. Largely ignored and distinguished by lack of primary sequence conservation, the linker is presumed to be intrinsically disordered. Here we employ genetics, biochemistry and cytology to dissect the role of the linker in FtsZ function. Data from chimeric FtsZs substituting the native linker with sequences from unrelated FtsZs as well as a helical sequence from human beta‐catenin indicate that while variations in the primary sequence are well tolerated, an intrinsically disordered linker is essential for Bacillus subtilis FtsZ assembly. Linker lengths ranging from 25 to 100 residues supported FtsZ assembly, but replacing the B. subtilis FtsZ linker with a 249‐residue linker from Agrobacterium tumefaciens FtsZ interfered with cell division. Overall, our results support a model in which the linker acts as a flexible tether allowing FtsZ to associate with the membrane through a conserved C‐terminal domain while simultaneously interacting with itself and modulatory proteins in the cytoplasm.     

Alanine and proline content modulate global sensitivity to discrete perturbations in disordered proteins

Molecular transduction of biological signals is understood primarily in terms of the cooperative structural transitions of protein macromolecules, providing a mechanism through which discrete local structure perturbations affect global macromolecular properties. The recognition that proteins lacking tertiary stability, commonly referred to as intrinsically disordered proteins (IDPs), mediate key signaling pathways suggests that protein structures without cooperative intramolecular interactions may also have the ability to couple local and global structure changes. Presented here are results from experiments that measured and tested the ability of disordered proteins to couple local changes in structure to global changes in structure. Using the intrinsically disordered N‐terminal region of the p53 protein as an experimental model, a set of proline (PRO) and alanine (ALA) to glycine (GLY) substitution variants were designed to modulate backbone conformational propensities without introducing non‐native intramolecular interactions. The hydrodynamic radius (R_h) was used to monitor changes in global structure. Circular dichroism spectroscopy showed that the GLY substitutions decreased polyproline II (PP_II) propensities relative to the wild type, as expected, and fluorescence methods indicated that substitution‐induced changes in R_h were not associated with folding. The experiments showed that changes in local PP_II structure cause changes in R_h that are variable and that depend on the intrinsic chain propensities of PRO and ALA residues, demonstrating a mechanism for coupling local and global structure changes. Molecular simulations that model our results were used to extend the analysis to other proteins and illustrate the generality of the observed PRO and alanine effects on the structures of IDPs. Proteins 2014; 82:3373–3384. © 2014 Wiley Periodicals, Inc.     

The intrinsically disordered linker of E. coli SSB is critical for the release from single‐stranded DNA

The Escherichia coli single stranded DNA binding protein (SSB) is crucial for DNA replication, recombination and repair. Within each process, it has two seemingly disparate roles: it stabilizes single‐stranded DNA (ssDNA) intermediates generated during DNA processing and, forms complexes with a group of proteins known as the SSB‐interactome. Key to both roles is the C‐terminal, one‐third of the protein, in particular the intrinsically disordered linker (IDL). Previously, they have shown using a series of linker deletion mutants that the IDL links both ssDNA and target protein binding by mediating interactions with the oligosaccharide/oligonucleotide binding fold in the target. In this study, they examine the role of the linker region in SSB function in a variety of DNA metabolic processes in vitro. Using the same linker mutants, the results show that in addition to association reactions (either DNA or protein), the IDL is critical for the release of SSB from DNA. This release can be under conditions of ssDNA competition or active displacement by a DNA helicase or recombinase. Consistent with their previous work these results indicate that SSB linker mutants are defective for SSB–SSB interactions, and when the IDL is removed a terminal SSB–DNA complex results. Formation of this complex inhibits downstream processing of DNA by helicases such as RecG or PriA as well as recombination, mediated by RecA. A model, based on the evidence herein, is presented to explain how the IDL acts in SSB function.     

Ensemble models of proteins and protein domains based on distance distribution restraints

Conformational ensembles of intrinsically disordered peptide chains are not fully determined by experimental observations. Uncertainty due to lack of experimental restraints and due to intrinsic disorder can be distinguished if distance distributions restraints are available. Such restraints can be obtained from pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy applied to pairs of spin labels. Here, we introduce a Monte Carlo approach for generating conformational ensembles that are consistent with a set of distance distribution restraints, backbone dihedral angle statistics in known protein structures, and optionally, secondary structure propensities or membrane immersion depths. The approach is tested with simulated restraints for a terminal and an internal loop and for a protein with 69 residues by using sets of sparse restraints for underlying well‐defined conformations and for published ensembles of a premolten globule‐like and a coil‐like intrinsically disordered protein. Proteins 2016; 84:544–560. © 2016 Wiley Periodicals, Inc.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural adaptation of tooth enamel protein amelogenin in the presence of SDS micelles

Amelogenin, the major extracellular matrix protein of developing tooth enamel is intrinsically disordered. Through its interaction with other proteins and mineral, amelogenin assists enamel biomineralization by controlling the formation of highly organized enamel crystal arrays. We used circular dichroism (CD), dynamic light scattering (DLS), fluorescence, and NMR spectroscopy to investigate the folding propensity of recombinant porcine amelogenin rP172 following its interaction with SDS, at levels above critical micelle concentration. The rP172‐SDS complex formation was confirmed by DLS, while an increase in the structure moiety of rP172 was noted through CD and fluorescence experiments. Fluorescence quenching analyses performed on several rP172 mutants where all but one Trp was replaced by Tyr at different sequence regions confirmed that the interaction of amelogenin with SDS micelles occurs via the N‐terminal region close to Trp25 where helical segments can be detected by NMR. NMR spectroscopy and structural refinement calculations using CS‐Rosetta modeling confirm that the highly conserved N‐terminal domain is prone to form helical structure when bound to SDS micelles. Our findings reported here reveal interactions leading to significant changes in the secondary structure of rP172 upon treatment with SDS. These interactions may reflect the physiological relevance of the flexible nature of amelogenin and its sequence specific helical propensity that might enable it to structurally adapt with charged and potential targets such as cell surface, mineral, and other proteins during enamel biomineralization. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 525–535, 2014.     

1H, 13C, and 15N resonance assignment of a 179 residue fragment of hepatitis C virus non-structural protein 5A

Non-structural protein 5A (NS5A) plays an important role in the life cycle of hepatitis C virus. This proline-rich phosphoprotein is organized into three domains. Besides its role in virus replication and virus assembly, NS5A is involved in a variety of cellular regulation processes. Recent studies on domain 2 and 3 revealed that both belong to the class of intrinsically disordered proteins as they adopt a natively unfolded state. In particular, domain 2 together with its vicinal regions is responsible for NS5A’s multiple interactions with other proteins necessary for virus persistence. The low chemical shift dispersion observed for instrinsically disordered proteins presents a challenge for NMR spectroscopy. Here we report sequential resonance assignment of a 179-residue fragment of NS5A, comprising the entire domain 2, using a set of sensitivity and resolution optimized 3D correlation experiments, as well as amino-acid-type editing in ¹H-¹⁵N correlation spectra. Our assignment reveals the presence of several segments with high propensity to form α-helical structure that may be of importance to the function of this protein fragment as a versatile interaction platform.     

Conformational and aggregation properties of the 1–93 fragment of apolipoprotein A‐I

Several disease‐linked mutations of apolipoprotein A‐I, the major protein in high‐density lipoprotein (HDL), are known to be amyloidogenic, and the fibrils often contain N‐terminal fragments of the protein. Here, we present a combined computational and experimental study of the fibril‐associated disordered 1–93 fragment of this protein, in wild‐type and mutated (G26R, S36A, K40L, W50R) forms. In atomic‐level Monte Carlo simulations of the free monomer, validated by circular dichroism spectroscopy, we observe changes in the position‐dependent β‐strand probability induced by mutations. We find that these conformational shifts match well with the effects of these mutations in thioflavin T fluorescence and transmission electron microscopy experiments. Together, our results point to molecular mechanisms that may have a key role in disease‐linked aggregation of apolipoprotein A‐I.     

The acidic domain is a unique structural feature of the splicing factor SYNCRIP

The splicing factor SYNCRIP (hnRNP Q) is involved in viral replication, neural morphogenesis, modulation of circadian oscillation, and the regulation of the cytidine deaminase APOBEC1. It consists of three globular RNA‐recognition motifs (RRM) domains flanked by an N‐terminal acid‐rich acidic sequence segment domain (AcD_12–97) and a C‐terminal domain containing an arginine–glycine‐rich sequence motif (RGG/RXG box), which are located near to the N‐ and C‐terminals, respectively. The acid‐rich sequence segment is unique to SYNCRIP and the closely related protein hnRNP R, and is involved in interactions with APOBEC1. Here, we show that while AcD_12–97 does not form a globular domain, structure‐based annotation identified a self‐folding globular domain with an all α‐helix architecture, AcD_24–107. The NMR structure of AcD_24?107 is fundamentally different from previously reported AcD molecular models. In addition to negatively charged surface areas, it contains a large hydrophobic cavity and a positively charged surface area as potential epitopes for intermolecular interactions.     

The C‐terminal calcium‐sensitive disordered motifs regulate isoform‐specific polymerization characteristics of calsequestrin

Calsequestrin (CASQ) exists as two distinct isoforms CASQ1 and CASQ2 in all vertebrates. Although the isoforms exhibit unique functional characteristic, the structural basis for the same is yet to be fully defined. Interestingly, the C‐terminal region of the two isoforms exhibit significant differences both in length and amino acid composition; forming Dn‐motif and DEXn‐motif in CASQ1 and CASQ2, respectively. Here, we investigated if the unique C‐terminal motifs possess Ca²⁺‐sensitivity and affect protein function. Sequence analysis shows that both the Dn‐ and DEXn‐motifs are intrinsically disordered regions (IDRs) of the protein, a feature that is conserved from fish to man. Using purified synthetic peptides, we show that these motifs undergo distinctive Ca²⁺‐mediated folding suggesting that these disordered motifs are Ca²⁺‐sensitivity. We generated chimeric proteins by swapping the C‐terminal portions between CASQ1 and CASQ2. Our studies show that the C‐terminal portions do not play significant role in protein folding. An interesting finding of the current study is that the switching of the C‐terminal portion completely reverses the polymerization kinetics. Collectively, these data suggest that these Ca²⁺‐sensitivity IDRs located at the back‐to‐back dimer interface influence isoform‐specific Ca²⁺‐dependent polymerization properties of CASQ. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 15–22, 2015.