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.     

A Large Intrinsically Disordered Region in SKIP and Its Disorder-Order Transition Induced by PPIL1 Binding Revealed by NMR

Intrinsically disordered proteins or protein regions play an important role in fundamental biological processes. During spliceosome activation, a large structural rearrangement occurs. The Prp19 complex and related factors are involved in the catalytic activation of the spliceosome. Recent mass spectrometric analyses have shown that Ski interaction protein (SKIP) and peptidylprolyl isomerase-like protein 1 (PPIL1) are Prp19-related factors that constitute the spliceosome B, B*, and C complexes. Here, we report that a highly flexible region of SKIP (SKIPN, residues 59–129) is intrinsically disordered. Upon binding to PPIL1, SKIPN undergoes a disorder-order transition. A highly conserved fragment of SKIP (residues 59–79) called the PPIL1-binding fragment (PBF) was sufficient to bind PPIL1. The structure of PBF·PPIL1 complex, solved by NMR, shows that PBF exhibits an ordered structure and interacts with PPIL1 through electrostatic and hydrophobic interactions. Three subfragments in the PBF (residues 59–67, 68–73, and 74–79) show hook-like backbone structure, and interactions between these subfragments are necessary for PBF·PPIL1 complex formation. PPIL1 is a cyclophilin family protein. It is recruited by SKIP into the spliceosome by a region other than the peptidylprolyl isomerase active site. This enables the active site of PPIL1 to remain open in the complex and still function as a peptidylprolyl cis/trans-isomerase or molecular chaperon to facilitate the folding of other proteins in the spliceosomes. The large disordered region in SKIP provides an interaction platform. Its disorder-order transition, induced by PPIL1 binding, may adapt the requirement for a large structural rearrangement occurred in the activation of spliceosome.     

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.     

Mutational and Cysteine Scanning Analysis of the Glucagon Receptor N-terminal Domain

The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp⁶³, Arg¹¹⁶, and Lys⁹⁸ are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp⁶³ and Arg¹¹⁶ form a salt bridge, whereas Lys⁹⁸ is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.     

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.     

The N-terminal Region of the Atypical Chemokine Receptor ACKR2 Is a Key Determinant of Ligand Binding

The atypical chemokine receptor, ACKR2 is a pivotal regulator of chemokine-driven inflammatory responses and works by binding, internalizing, and degrading inflammatory CC-chemokines. ACKR2 displays promiscuity of ligand binding and is capable of interacting with up to 14 different inflammatory CC-chemokines. Despite its prominent biological role, little is known about the structure/function relationship within ACKR2, which regulates ligand binding. Here we demonstrate that a conserved tyrosine motif at the N terminus of ACKR2 is essential for ligand binding, internalization, and scavenging. In addition we demonstrate that sulfation of this motif contributes to ligand internalization. Furthermore, a peptide derived from this region is capable of binding inflammatory chemokines and inhibits their interaction with their cognate signaling receptors. Importantly, the peptide is only active in the sulfated form, further confirming the importance of the sulfated tyrosines for function. Finally, we demonstrate that the bacterial protease, staphopain A, can cleave the N terminus of ACKR2 and suppress its ligand internalization activity. Overall, these results shed new light on the nature of the structural motifs in ACKR2 that are responsible for ligand binding. The study also highlights ACKR2-derived N-terminal peptides as being of potential therapeutic significance.     

Third Transmembrane Domain of the Adrenocorticotropic Receptor Is Critical for Ligand Selectivity and Potency

The ACTH receptor, known as the melanocortin-2 receptor (MC2R), plays an important role in regulating and maintaining adrenocortical function. MC2R is a subtype of the melanocortin receptor (MCR) family and has unique characteristics among MCRs. Endogenous ACTH is the only endogenous agonist for MC2R, whereas the melanocortin peptides α-, β-, and γ-melanocyte-stimulating hormone and ACTH are full agonists for all other MCRs. In this study, we examined the molecular basis of MC2R responsible for ligand selectivity using ACTH analogs and MC2R mutagenesis. Our results indicate that substitution of Phe⁷ with d-Phe or d-naphthylalanine (d-Nal(2′)) in ACTH(1–24) caused a significant decrease in ligand binding affinity and potency. Substitution of Phe⁷ with d-Nal(2′) in ACTH(1–24) did not switch the ligand from agonist to antagonist at MC2R, which was observed in MC3R and MC4R. Substitution of Phe⁷ with d-Phe⁷ in ACTH(1–17) resulted in the loss of ligand binding and activity. Molecular analysis of MC2R indicated that only mutation of the third transmembrane domain of MC2R resulted in a decrease in d-Phe ACTH binding affinity and potency. Our results suggest that Phe⁷ in ACTH plays an important role in ligand selectivity and that the third transmembrane domain of MC2R is crucial for ACTH selectivity and potency.     

Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain

A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2_hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97–125 + Tyr), interacted specifically with isolated K2_hPg. However, the binding strength of VEK30 (K_D = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (K_D = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH₂ and COOH termini of VEK30. VEK64 (PAM residues 83–145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2_hPg with nearly the same affinity as PAM (K_D = 1–2 nm). However, addition of two PAM residues (Arg¹²⁶-His¹²⁷) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2_hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg¹¹³, His¹¹⁴, Glu¹¹⁶, Arg¹²⁶, His¹²⁷), mutation of which reduced PAM binding affinity for K2_hPg by ∼1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence.     

Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy-Terminal Region, but Is Not Caused by Receptor Internalization

Abstract: The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 µ M neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 n M ) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µ M substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn²⁺ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.