Interaction of nonstructural protein 5A of the hepatitis C virus with Src homology 3 domains using noncanonical binding sites.

Src homology 3 (SH3) domains are widely known for their ability to interact with other proteins using the canonical PxxP binding motif. Besides those well-characterized interaction modes, there is an increasing number of SH3 domain-containing complexes that lack this motif. Here we characterize the interaction of SH3 domains, in particular the Bin1-SH3 domain, with the intrinsically disordered part of nonstructural protein 5A of the hepatitis C virus using noncanonical binding sites in addition to its PxxP motif. These binding regions partially overlap with regions that have previously been identified as having an increased propensity to form α-helices. Remarkably, upon interaction with the Bin1-SH3 domain, the α-helical propensity decreases and a fuzzy complex is formed.

Helix formation in arrestin accompanies recognition of photoactivated rhodopsin.

Binding of arrestin to photoactivated phosphorylated rhodopsin terminates the amplification of visual signals in photoreceptor cells. Currently, there is no crystal structure of a rhodopsin-arrestin complex available, although structures of unbound rhodopsin and arrestin have been determined. High-affinity receptor binding is dependent on distinct arrestin sites responsible for recognition of rhodopsin activation and phosphorylation. The loop connecting beta-strands V and VI in rod arrestin has been implicated in the recognition of active rhodopsin. We report the structure of receptor-bound arrestin peptide Arr(67-77) mimicking this loop based on solution NMR data. The peptide binds photoactivated rhodopsin in the unphosphorylated and phosphorylated form with similar affinities and stabilizes the metarhodopsin II photointermediate. A largely alpha-helical conformation of the receptor-bound peptide is observed.

Integral membrane proteins in nanodiscs can be studied by solution NMR spectroscopy.

We present a two-dimensional solution NMR spectrum of an integral membrane protein (IMP) in a nanodisc. Solution NMR relies on rapid isotropic tumbling of the analyte with correlation times in the nanosecond range. IMPs in a cellular membrane do not satisfy this condition. Previous liquid-state NMR studies on IMPs were conducted in organic solvent or artificial membrane mimicking particles like detergent micelles. Nanodiscs are relatively small (150 kDa), detergent-free model membranes that are suitable for functional reconstitution of IMPs. Nanodiscs allow solubilization of integral membrane proteins in a nearly native lipid bilayer environment. The 70 residue polypeptide CD4mut was incorporated into nanodiscs. CD4mut features one transmembrane helix. The aliphatic (1)H-(13)C HSQC spectrum of nanodiscs with inserted, ((13)C, (15)N)-labeled CD4mut exhibits reasonably dispersed protein and lipid NMR signals. Our results demonstrate that IMPs in nanodiscs are amenable to liquid-state NMR methodology.

Zika virus NS2B/NS3 proteinase: A new target for an old drug - Suramin a lead compound for NS2B/NS3 proteinase inhibition.

Zika virus infection is the focus of much research due to the medical and social repercussions. Due the role of the viral NS2B/NS3 proteinase in maturation of the viral proteins, it had become an attractive antiviral target. Numerous investigations on viral epidemiology, structure and function analysis, vaccines, and therapeutic drugs have been conducted around the world. At present, no approved vaccine or even drugs have been reported. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified the polyanion suramin, an approved antiparasitic drug with antiviral properties, as a potential inhibitor of Zika virus complex NS2B/NS3 proteinase with IC50 of 47 µM. Using fluorescence spectroscopy results we could determine a kd value of 28 µM and had shown that the ligand does not affect the thermal stability of the protein. STD NMR spectroscopy experiments and molecular docking followed by molecular dynamics simulation identified the binding epitopes of the molecule and shows the mode of interaction, respectively. The computational analysis showed that suramin block the Ser135 residue and interact with the catalytically histidine residue.

ß-Hairpin of Islet Amyloid Polypeptide Bound to an Aggregation Inhibitor.

In type 2 diabetes, the formation of islet amyloid consisting of islet amyloid polypeptide (IAPP) is associated with reduction in ß-cell mass and contributes to the failure of islet cell transplantation. Rational design of inhibitors of IAPP amyloid formation has therapeutic potential, but is hampered by the lack of structural information on inhibitor complexes of the conformationally flexible, aggregation-prone IAPP. Here we characterize a ß-hairpin conformation of IAPP in complex with the engineered binding protein ß-wrapin HI18. The ß-strands correspond to two amyloidogenic motifs, 12-LANFLVH-18 and 22-NFGAILS-28, which are connected by a turn established around Ser-20. Besides backbone hydrogen bonding, the IAPP:HI18 interaction surface is dominated by non-polar contacts involving hydrophobic side chains of the IAPP ß-strands. Apart from monomers, HI18 binds oligomers and fibrils and inhibits IAPP aggregation and toxicity at low substoichiometric concentrations. The IAPP ß-hairpin can serve as a molecular recognition motif enabling control of IAPP aggregation.

iHADAMAC: a complementary tool for sequential resonance assignment of globular and highly disordered proteins.

An experiment, iHADAMAC, is presented that yields information on the amino-acid type of individual residues in a protein by editing the (1)H-(15)N correlations into seven different 2D spectra, each corresponding to a different class of amino-acid types. Amino-acid type discrimination is realized via a Hadamard encoding scheme based on four different spin manipulations as recently introduced in the context of the sequential HADAMAC experiment. Both sequential and intra-residue HADAMAC experiments yield highly complementary information that greatly facilitate resonance assignment of proteins with high frequency degeneracy, as demonstrated here for a 188-residue intrinsically disordered protein fragment of the hepatitis C virus protein NS5A.

Transient structure and SH3 interaction sites in an intrinsically disordered fragment of the hepatitis C virus protein NS5A.

Understanding the molecular mechanisms involved in virus replication and particle assembly is of primary fundamental and biomedical importance. Intrinsic conformational disorder plays a prominent role in viral proteins and their interaction with other viral and host cell proteins via transiently populated structural elements. Here, we report on the results of an investigation of an intrinsically disordered 188-residue fragment of the hepatitis C virus non-structural protein 5A (NS5A), which contains a classical poly-proline Src homology 3 (SH3) binding motif, using sensitivity- and resolution-optimized multidimensional NMR methods, complemented by small-angle X-ray scattering data. Our study provides detailed atomic-resolution information on transient local and long-range structure, as well as fast time scale dynamics in this NS5A fragment. In addition, we could characterize two distinct interaction modes with the SH3 domain of Bin1 (bridging integrator protein 1), a pro-apoptotic tumor suppressor. Despite being largely disordered, the protein contains three regions that transiently adopt α-helical structures, partly stabilized by long-range tertiary interactions. Two of these transient α-helices form a noncanonical SH3-binding motif, which allows low-affinity SH3 binding. Our results contribute to a better understanding of the role of the NS5A protein during hepatitis C virus infection. The present work also highlights the power of NMR spectroscopy to characterize multiple binding events including short-lived transient interactions between globular and highly disordered proteins.

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 (1)H-(15)N 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.

Competitively selected protein ligands pay their increase in specificity by a decrease in affinity.

Protein-ligand interactions characterise and govern the current state and fate of a living cell. The specificity of proteins is mainly determined by the relative affinities to each potential ligand. To investigate the consequences and potentials of ligands with increased specificity in comparison with ligands optimised solely for affinity, it was necessary to identify ligands that are optimised towards specificity instead of a barely optimised affinity to a given target. In the presented example, a modified phage display screening procedure yielded specific ligands for the LckSH3 domain. We found that increased specificity of one of the hereby obtained ligands for LckSH3 is achieved at the cost of a slightly reduced affinity to LckSH3 and a drastically reduced affinity to other SH3 domains. A surface plasmon resonance experiment simulating in vivo-like realistic competitive binding conditions exerted enhanced binding behaviour of the specific ligand under these binding conditions. The experimental data, together with a mathematical model describing the complex experimental situation, and theoretical considerations lead to the conclusion that increased specificity is achieved at the cost of reduced affinity, but after all, it pays if the ligand is applied under realistic, i.e. competitive, conditions.

Nonaqueous versus aqueous capillary electrophoresis of alpha-helical polypeptides: effect of secondary structure on separation selectivity.

The CE separation of alpha-helical polypeptides composed of 14-31 amino acid residues has been investigated using aqueous and nonaqueous BGEs. The running buffers were optimized with respect to pH. Generally, higher separation selectivities were observed in nonaqueous electrolytes. This may be explained by a change in the secondary structure when changing from water to organic solvents. Circular dichroism spectra revealed a significant increase in helical structures in methanol-based buffers compared to aqueous buffers. This change in secondary structure of the polypeptides contributed primarily to the different separation selectivity observed in aqueous CE and NACE. For small oligopeptides of two to five amino acid residues no significant effect of the solvent was observed in some cases while in other cases a reversal of the migration order occurred when changing from aqueous to nonaqueous buffers. As these peptides cannot adopt secondary structures the effect may be attributed to a shift of the pKa values in organic solvents compared to water.

Prevention of Alzheimer's disease-associated Abeta aggregation by rationally designed nonpeptidic beta-sheet ligands.

A new concept is introduced for the rational design of beta-sheet ligands, which prevent protein aggregation. Oligomeric acylated aminopyrazoles with a donor-acceptor-donor (DAD) hydrogen bond pattern complementary to that of a beta-sheet efficiently block the solvent-exposed beta-sheet portions in Abeta-(1-40) and thereby prevent formation of insoluble protein aggregates. Density gradient centrifugation revealed that in the initial phase, the size of Abeta aggregates was efficiently kept between the trimeric and 15-meric state, whereas after 5 days an additional high molecular weight fraction appeared. With fluorescence correlation spectroscopy (FCS) exactly those two, i.e. a dimeric aminopyrazole with an oxalyl spacer and a trimeric head-to-tail connected aminopyrazole, of nine similar aminopyrazole ligands were identified as efficient aggregation retardants whose minimum energy conformations showed a perfect complementarity to a beta-sheet. The concentration dependence of the inhibitory effect of a trimeric aminopyrazole derivative allowed an estimation of the dissociation constant in the range of 10(-5) m. Finally, electrospray ionization mass spectrometry (ESI-MS) was used to determine the aggregation kinetics of Abeta-(1-40) in the absence and in the presence of the ligands. From the comparable decrease in Abeta monomer concentration, we conclude that these beta-sheet ligands do not prevent the initial oligomerization of monomeric Abeta but rather block further aggregation of spontaneously formed small oligomers. Together with the results from density gradient centrifugation and fluorescence correlation spectroscopy it is now possible to restrict the approximate size of soluble Abeta aggregates formed in the presence of both inhibitors from 3- to 15-mers.