The novel fold of scytovirin reveals a new twist for antiviral entry inhibitors

The solution structure of the potent 95 residue anti-HIV protein scytovirin has been determined and two carbohydrate-binding sites have been identified. This unique protein, containing five structurally important disulfide bonds, demonstrates a novel fold with no elements of extended regular secondary structure. Scytovirin contains two 39 residue sequence repeats, differing in only three amino acid residues, and each repeat has primary sequence similarity to chitin binding proteins. Both sequence repeats form similarly structured domains, with the exception of one region. The result is two carbohydrate-binding sites with substantially different affinities. The unusual fold clusters aromatic residues in both sites, suggesting a binding mechanism similar to other known hevein-like carbohydrate-binding proteins but differing in carbohydrate specificity. Scytovirin, originally isolated from the cyanobacterium Scytonema varium, holds potential as an HIV entry inhibitor for both therapeutic and prophylactic anti-HIV applications. The high-resolution structural studies reported are an important initial step in unlocking the therapeutic potential of scytovirin.     

Programmed ribosomal frameshifting in SIV is induced by a highly structured RNA stem-loop

Simian immunodeficiency virus (SIV), like its human homologues (HIV-1, HIV-2), requires a -1 translational frameshift event to properly synthesize all of the proteins required for viral replication. The frameshift mechanism is dependent upon a seven-nucleotide slippery sequence and a downstream RNA structure. In SIV, the downstream RNA structure has been proposed to be either a stem-loop or a pseudoknot. Here, we report the functional, structural and thermodynamic characterization of the SIV frameshift site RNA. Translational frameshift assays indicate that a stem-loop structure is sufficient to promote efficient frameshifting in vitro. NMR and thermodynamic studies of SIV RNA constructs of varying length further support the absence of any pseudoknot interaction and indicate the presence of a stable stem-loop structure. We determined the structure of the SIV frameshift-inducing RNA by NMR. The structure reveals a highly ordered 12 nucleotide loop containing a sheared G-A pair, cross-strand adenine stacking, two G-C base-pairs, and a novel CCC triloop turn. The loop structure and its high thermostability preclude pseudoknot formation. Sequence conservation and modeling studies suggest that HIV-2 RNA forms the same structure. We conclude that, like the main sub-groups of HIV-1, SIV and HIV-2 utilize stable stem-loop structures to function as a thermodynamic barrier to translation, thereby inducing ribosomal pausing and frameshifting.     

A Method for Helical RNA Global Structure Determination in Solution Using Small-Angle X-Ray Scattering and NMR Measurements

We report a “top-down” method that uses mainly duplexes' global orientations and overall molecular dimension and shape restraints, which were extracted from experimental NMR and small-angle X-ray scattering data, respectively, to determine global architectures of RNA molecules consisting of mostly A-form-like duplexes. The method is implemented in the G2G (from global measurement to global structure) toolkit of programs. We demonstrate the efficiency and accuracy of the method by determining the global structure of a 71-nt RNA using experimental data. The backbone root-mean-square deviation of the ensemble of the calculated global structures relative to the X-ray crystal structure is 3.0 ± 0.3 Å using the experimental data and is only 2.5 ± 0.2 Å for the three duplexes that were orientation restrained during the calculation. The global structure simplifies interpretation of multidimensional nuclear Overhauser spectra for high-resolution structure determination. The potential general application of the method for RNA structure determination is discussed.     

Folding Topology of a Bimolecular DNA Quadruplex Containing a Stable Mini-hairpin Motif within the Diagonal Loop

We describe the NMR structural characterisation of a bimolecular anti-parallel DNA quadruplex d(G₃ACGTAGTG₃)₂ containing an autonomously stable mini-hairpin motif inserted within the diagonal loop. A folding topology is identified that is different from that observed for the analogous d(G₃T₄G₃)₂ dimer with the two structures differing in the relative orientation of the diagonal loops. This appears to reflect specific base stacking interactions at the quadruplex-duplex interface that are not present in the structure with the T₄-loop sequence. A truncated version of the bimolecular quadruplex d(G₂ACGTAGTG₂)₂, with only two core G-tetrads, is less stable and forms a heterogeneous mixture of three 2-fold symmetric quadruplexes with different loop arrangements. We demonstrate that the nature of the loop sequence, its ability to form autonomously stable structure, the relative stabilities of the hairpin loop and core quadruplex, and the ability to form favourable stacking interactions between these two motifs are important factors in controlling DNA G-quadruplex topology.     

The cytoplasmic domain of the chloride channel ClC-0: structural and dynamic characterization of flexible regions

Eukaryotic members of the ClC family of chloride channels and transporters are composed of a transmembrane ion transport domain followed by a cytoplasmic domain, which is believed to be involved in the modulation of ClC function. In some family members this putative regulatory domain contains next to a well-folded structured part, long sequence stretches with low sequence complexity. These regions, a 96 residue long linker connecting two structured sub-domains, and 35 residues on the C teminus of the domain were found disordered in a recent crystal structure of this domain in ClC-0. Both regions have a large influence on the modulation of channel function in closely related family members. Here we describe a NMR study to characterize the structural and dynamic properties of these putatively unstructured stretches. Our study reveals that the two regions indeed show large conformational flexibility with dynamics on the nanosecond timescale. However, small islands of secondary structure are found interdispersed between the unfolded regions. This study characterizes for the first time the biophysical properties of these protein segments, which may become important for the understanding of novel regulatory mechanisms within the ClC family.     

Solution Structure of S100A1 Bound to the CapZ Peptide (TRTK12)

As is typical for S100-target protein interactions, a Ca²⁺-dependent conformational change in S100A1 is required to bind to a 12-residue peptide (TRTK12) derived from the actin-capping protein CapZ. In addition, the Ca²⁺-binding affinity of S100A1 is found to be tightened (greater than threefold) when TRTK12 is bound. To examine the biophysical basis for these observations, we determined the solution NMR structure of TRTK12 in a complex with Ca²⁺-loaded S100A1. When bound to S100A1, TRTK12 forms an amphipathic helix (residues N6 to S12) with several favorable hydrophobic interactions observed between W7, I10, and L11 of the peptide and a well-defined hydrophobic binding pocket in S100A1 that is only present in the Ca²⁺-bound state. Next, the structure of S100A1-TRTK12 was compared to that of another S100A1-target complex (i.e., S100A1-RyRP12), which illustrated how the binding pocket in Ca²⁺-S100A1 can accommodate peptide targets with varying amino acid sequences. Similarities and differences were observed when the structures of S100A1-TRTK12 and S100B-TRTK12 were compared, providing insights regarding how more than one S100 protein can interact with the same peptide target. Such comparisons, including those with other S100-target and S100-drug complexes, provide the basis for designing novel small-molecule inhibitors that could be specific for blocking one or more S100-target protein interactions.     

The solution and crystal structures of a module pair from the Staphylococcus aureus-binding site of human fibronectin--a tale with a twist

An important goal of structural studies of modular proteins is to determine the inter-module orientation, which often influences biological function. The N-terminal domain of human fibronectin (Fn) is composed of a string of five type 1 modules (F1). Despite their small size, to date F1 modules have proved intractable to X-ray structure solution, although there are several NMR structures available. Here, we present the first structures (two X-ray models and an NMR-derived model) of the (2)F1(3)F1 module pair, which forms part of the binding site for Fn-binding proteins from pathogenic bacteria. The crystallographic structure determination was aided by the novel technique of UV radiation damage-induced phasing. The individual module structures are very similar in all three models. In the NMR structure and one of the X-ray structures, a similar but smaller interdomain interface than that observed previously for (4)F1(5)F1 is seen. The other X-ray structure has a different interdomain orientation. This work underlines the benefits of combining X-ray and NMR data in the studies of multi-domain proteins.     

An Unusual Mode of Galactose Recognition by a Family 32 Carbohydrate-Binding Module

Carbohydrate-binding modules (CBMs) are ancillary modules commonly associated with carbohydrate-active enzymes (CAZymes) that function to mediate the adherence of the parent enzyme to its carbohydrate substrates. CBM family 32 (CBM32) is one of the most diverse CBM families, whose members are commonly found in bacterial CAZymes that modify eukaryotic glycans. One such example is the putative μ-toxin, CpGH84A, of the family 84 glycoside hydrolases, which comprises an N-terminal putative β-N-acetylglucosaminidase catalytic module and four tandem CBM32s. Here, we report a unique mode of galactose recognition by the first CBM32, CBM32-1 from CpGH84A. Solution NMR-based analyses of CpGH84A CBM32-1 indicate a divergent subset of residues, located in ordered loops at the apex of the CBM, conferring specificity for the galacto-configured sugars galactose, GalNAc, and LacNAc that differs from those of the canonical galactose-binding CBM32s. This study showcases the impressive variability in ligand binding by this CBM family and offers insight into the growing role of these modules in the interaction of CAZymes with eukaryotic glycans.     

Proline Isomerization Preorganizes the Itk SH2 Domain for Binding to the Itk SH3 Domain

We report here the NMR-derived structure of the binary complex formed by the interleukin-2 tyrosine kinase (Itk) Src homology 3 (SH3) and Src homology 2 (SH2) domains. The interaction is independent of both a phosphotyrosine motif and a proline-rich sequence, the classical targets of the SH2 and SH3 domains, respectively. The Itk SH3/SH2 structure reveals the molecular details of this nonclassical interaction and provides a clear picture for how the previously described prolyl cis/trans isomerization present in the Itk SH2 domain mediates SH3 binding. The higher-affinity cis SH2 conformer is preorganized to form a hydrophobic interface with the SH3 domain. The structure also provides insight into how autophosphorylation in the Itk SH3 domain might increase the affinity of the intermolecular SH3/SH2 interaction. Finally, we can compare this Itk complex with other examples of SH3 and SH2 domains engaging their ligands in a nonclassical manner. These small binding domains exhibit a surprising level of diversity in their binding repertoires.     

The solution structure of antigen MPT64 from Mycobacterium tuberculosis defines a new family of beta-grasp proteins

The MPT64 protein and its homologs form a highly conserved family of secreted proteins with unknown function that are found within the pathogenic Mycobacteria genus. The founding member of this family from Mycobacterium tuberculosis (MPT64 or protein Rv1980c) is expressed only when Mycobacteria cells are actively dividing. By virtue of this relatively unique expression profile, Rv1980c is currently under phase III clinical trials to evaluate its potential to replace tuberculin, or purified protein derivative, as the rapid diagnostic of choice for detection of active tuberculosis infection. We describe here the NMR solution structure of Rv1980c. This structure reveals a previously undescribed fold that is based upon a variation of a beta-grasp motif most commonly found in protein-protein interaction domains. Examination of this structure in conjunction with multiple sequence alignments of MPT64 homologs identifies a candidate ligand-binding site, which may help guide future studies of Rv1980c function. The work presented here also suggests structure-based approaches for increasing the antigenic potency of a Rv1980c-based diagnostic.     

Structural Interpretation of Paramagnetic Relaxation Enhancement-Derived Distances for Disordered Protein States

Paramagnetic relaxation enhancement (PRE) is a powerful technique for studying transient tertiary organizations of unfolded and partially folded proteins. The heterogeneous and dynamic nature of disordered protein states, together with the r⁻⁶ dependence of PRE, presents significant challenges for reliable structural interpretation of PRE-derived distances. Without additional knowledge of accessible conformational substates, ensemble-simulation-based protocols have been used to calculate structure ensembles that appear to be consistent with the PRE distance restraints imposed on the ensemble level with the proper r⁻⁶ weighting. However, rigorous assessment of the reliability of such protocols has been difficult without intimate knowledge of the true nature of disordered protein states. Here we utilize sets of theoretical PRE distances derived from simulated structure ensembles that represent the folded, partially folded and unfolded states of a small protein to investigate the efficacy of ensemble-simulation-based structural interpretation of PRE distances. The results confirm a critical limitation that, due to r⁻⁶ weighting, only one or a few members need to satisfy the distance restraints and the rest of the ensemble are essentially unrestrained. Consequently, calculated structure ensembles will appear artificially heterogeneous no matter whether the PRE distances are derived from the folded, partially unfolded or unfolded state. Furthermore, the nature of the heterogeneous ensembles is largely determined by the protein model employed in structure calculation and reflects little on the true nature of the underlying disordered state. These findings suggest that PRE measurements on disordered protein states alone generally do not contain enough information for a reliable structural interpretation and that the latter will require additional knowledge of accessible conformational substates. Interestingly, when a very large number of PRE measurements is available, faithful structural interpretation might be possible with intermediate ensemble sizes under ideal conditions.     

The Conformational Properties of the Glc3Man Unit Suggest Conformational Biasing within the Chaperone-assisted Glycoprotein Folding Pathway

A major puzzle is: are all glycoproteins routed through the ER calnexin pathway irrespective of whether this is required for their correct folding? Calnexin recognizes the terminal Glcα1-3Manα linkage, formed by trimming of the Glcα1-2Glcα1-3Glcα1-3Manα (Glc₃Man) unit in Glc₃Man₉GlcNAc₂. Different conformations of this unit have been reported. We have addressed this problem by studying the conformation of a series of N-glycans; i.e. Glc₃ManOMe, Glc₃Man₄,₅,₇GlcNAc₂ and Glc₁Man₉GlcNAc₂ using 2D NMR NOESY, ROESY, T-ROESY and residual dipolar coupling experiments in a range of solvents, along with solution molecular dynamics simulations of Glc₃ManOMe. Our results show a single conformation for the Glcα1-2Glcα and Glcα1-3Glcα linkages, and a major (65%) and a minor (30%) conformer for the Glcα1-3Manα linkage. Modeling of the binding of Glc₁Man₉GlcNAc₂ to calnexin suggests that it is the minor conformer that is recognized by calnexin. This may be one of the mechanisms for controlling the rate of recruitment of proteins into the calnexin/calreticulin chaperone system and enabling proteins that do not require such assistance for folding to bypass the system. This is the first time evidence has been presented on glycoprotein folding that suggests the process may be optimized to balance the chaperone-assisted and chaperone-independent pathways.     

Structure of H/ACA RNP protein Nhp2p reveals cis/trans isomerization of a conserved proline at the RNA and Nop10 binding interface

H/ACA small nucleolar and Cajal body ribonucleoproteins (RNPs) function in site-specific pseudouridylation of eukaryotic rRNA and snRNA, rRNA processing, and vertebrate telomerase biogenesis. Nhp2, one of four essential protein components of eukaryotic H/ACA RNPs, forms a core trimer with the pseudouridylase Cbf5 and Nop10 that binds to H/ACA RNAs specifically. Crystal structures of archaeal H/ACA RNPs have revealed how the protein components interact with each other and with the H/ACA RNA. However, in place of Nhp2p, archaeal H/ACA RNPs contain L7Ae, which binds specifically to an RNA K-loop motif absent from eukaryotic H/ACA RNPs, while Nhp2 binds a broader range of RNA structures. We report solution NMR studies of Saccharomyces cerevisiae Nhp2 (Nhp2p), which reveal that Nhp2p exhibits two major conformations in solution due to cis/trans isomerization of the evolutionarily conserved Pro83. The equivalent proline is in the cis conformation in all reported structures of L7Ae and other homologous proteins. Nhp2p has the expected α-β-α fold, but the solution structures of the major conformation of Nhp2p with trans Pro83 and of Nhp2p-S82W with cis Pro83 reveal that Pro83 cis/trans isomerization affects the positions of numerous residues at the Nop10 and RNA binding interface. An S82W substitution, which stabilizes the cis conformation, also stabilizes the association of Nhp2p with H/ACA snoRNPs expressed in vivo. We propose that Pro83 plays a key role in the assembly of the eukaryotic H/ACA RNP, with the cis conformation locking in a stable Cbf5-Nop10-Nhp2 ternary complex and positioning the protein backbone to interact with the H/ACA RNA.     

Solution structures of human and porcine beta-microseminoprotein

Beta-microseminoprotein (MSP) is a small cysteine-rich protein (molecular mass about 10 kDa) first isolated from human seminal plasma and later identified in several other organisms. The function of MSP is not known, but a recent study has shown MSP to bind CRISP-3, a protein present in neutrophilic granulocytes. The amino acid sequence is highly variable between species raising the question of the evolutionary conservation of the 3D structure. Here we present NMR solution structures of both the human and the porcine MSP. The two proteins (sequence identity 51%) have a very similar 3D structure with the secondary structure elements well conserved and with most of the amino acid substitutions causing a change of charge localized to one side of the molecule. MSP is a beta-sheet-rich protein with two distinct domains. The N-terminal domain is composed of a four-stranded beta-sheet, with the strands arranged according to the Greek key-motif, and a less structured part. The C-terminal domain contains two two-stranded beta-sheets with no resemblance to known structural motifs. The two domains, connected to each other by the peptide backbone, one disulfide bond, and interactions between the N and C termini, are oriented to give the molecule a rather extended structure. This global fold differs markedly from that of a previously published structure for porcine MSP, in which the two domains have an entirely different orientation to each other. The difference probably stems from a misinterpretation of ten specific inter-domain NOEs.     

Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA

The zinc finger domain of the Wilms tumor suppressor protein (WT1) contains four canonical Cys(2)His(2) zinc fingers. WT1 binds preferentially to DNA sequences that are closely related to the EGR-1 consensus site. We report the structure determination by both X-ray crystallography and NMR spectroscopy of the WT1 zinc finger domain in complex with DNA. The X-ray structure was determined for the complex with a cognate 14 base-pair oligonucleotide, and composite X-ray/NMR structures were determined for complexes with both the 14 base-pair and an extended 17 base-pair DNA. This combined approach allowed unambiguous determination of the position of the first zinc finger, which is influenced by lattice contacts in the crystal structure. The crystal structure shows the second, third and fourth zinc finger domains inserted deep into the major groove of the DNA where they make base-specific interactions. The DNA duplex is distorted in the vicinity of the first zinc finger, with a cytidine twisted and tilted out of the base stack to pack against finger 1 and the tip of finger 2. By contrast, the composite X-ray/NMR structures show that finger 1 continues to follow the major groove in the solution complexes. However, the orientation of the helix is non-canonical, and the fingertip and the N terminus of the helix project out of the major groove; as a consequence, the zinc finger side-chains that are commonly involved in base recognition make no contact with the DNA. We conclude that finger 1 helps to anchor WT1 to the DNA by amplifying the binding affinity although it does not contribute significantly to binding specificity. The structures provide molecular level insights into the potential consequences of mutations in zinc fingers 2 and 3 that are associated with Denys-Drash syndrome and nephritic syndrome. The mutations are of two types, and either destabilize the zinc finger structure or replace key base contact residues.     

Enhanced stability of cis Pro-Pro peptide bond in Pro-Pro-Phe sequence motif

Identification of sequence motifs that favor cis peptide bonds in proteins is important for understanding and designing proteins containing turns mediated by cis peptide conformations. From (1)H NMR solution studies on short peptides, we show that the Pro-Pro peptide bond in Pro-Pro-Phe almost equally populates the cis and trans isomers, with the cis isomer stabilized by a CHc...pi interaction involving the terminal Pro and Phe. We also show that Phe is over-represented at sequence positions immediately following cis Pro-Pro motifs in known protein structures. Our results demonstrate that the Pro-Pro cis conformer in Pro-Pro-Phe sequence motifs is as important as the trans conformer, both in short peptides as well as in natively folded proteins.