New binding specificities derived from Min-23, a small cystine-stabilized peptidic scaffold

The randomization of both internal and surface residues in small protein domains followed by selection from a display library is emerging as a powerful strategy to obtain novel binding specificities. Small and stable scaffold motifs observed in disulfide-rich proteins are attractive because they are small, stable, and accessible to chemical synthesis. The elementary structural motif found in the squash trypsin inhibitor EETI-II (Ecballium elaterium trypsin inhibitor) is the cystine stabilized beta-sheet (CSB) motif, found in nearly 50% of all known small disulfide-rich protein families. We have used Min-23, a short 23-residue peptide containing the CSB motif and shown to be a stable autonomous folding unit and one of the smallest scaffolds described to date, as a scaffold for selection of new binding ligands. We demonstrate that the core CSB motif in Min-23 is permissive to loop insertion, using peptide epitopes from hemagglutinin (HA) and Gla-protein (E). A phage library of more than 10(8) different clones has been constructed by insertion of a randomized sequence on a beta-turn of the Min-23 peptide. The selection of this library on a variety of 7 different targets allowed the isolation of 21 new specific binders, confirming the potential of Min-23 as a scaffold for the development of new ligands. The derived library is able to provide a wide range of novel compounds with possible applications in various biological and pharmaceutical areas.     

Role of the tyrosine corner motif in the stability of neocarzinostatin

Although the immunoglobulin-like beta-sandwich fold has no specifically conserved function, some common structural features have been observed, in particular a structural motif, the tyrosine corner. Such a motif was described in neocarzinostatin (NCS), a bacterial protein the structure of which is very similar to that of the immunoglobulin domain. Compared with the other beta-sheet proteins, the NCS 'tyrosine corner' presents non-standard structural features. To investigate the role of this motif in the NCS structure and stability, we studied the properties of a mutant where the H bond interaction had been eliminated by replacing the tyrosine with a phenylalanine. This mutation costs 4.0 kcal/mol showing that the NCS 'tyrosine corner' is involved in protein stability as in the other Greek key proteins. This destabilization is accompanied by remote structural effects, including modification of the binding properties, suggesting an increase in the internal flexibility of the protein. With a view to using this protein for drug targeting, these results along with those obtained previously allow us to define clearly the limitations of the modifications that can be performed on this scaffold.     

Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif-like domain

We have determined the crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 at 1.9 A resolution. This protein is a member of the Escherichia coli ygcH sequence family, which contains approximately 15 sequence homologs of bacterial origin. These homologs have a high isoelectric point. The crystal structure reveals that TTHB192 consists of two independently folded domains, and that each domain exhibits a ferredoxin-like fold with a four-stranded antiparallel beta-sheet packed on one side by alpha-helices. These two tandem domains face each other to generate a beta-sheet platform. TTHB192 displays overall structural similarity to Sex-lethal protein and poly(A)-binding protein fragments. These proteins have RNA binding activity which is supported by a beta-sheet platform formed by two tandem repeats of an RNA recognition motif domain with signature sequence motifs on the beta-sheet surface. Although TTHB192 does not have the same signature sequence motif as the RNA recognition motif domain, the presence of an evolutionarily conserved basic patch on the beta-sheet platform could be functionally relevant for nucleic acid-binding. This report shows that TTHB192 and its sequence homologs adopt an RNA recognition motif-like domain and provides the first testable functional hypothesis for this protein family.     

The structure of spider toxin huwentoxin-II with unique disulfide linkage: Evidence for structural evolution

The three-dimensional structure of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of spider Selenocosmia huwena with a unique disulfide bond linkage as I-III, II-V, and IV-VI, has been determined using 2D (1)H-NMR. The resulting structure of HWTX-II contains two beta-turns (C4-S7 and K24-W27) and a double-stranded antiparallel beta-sheet (W27-C29 and C34-K36). Although the C-terminal double-stranded beta-sheet cross-linked by two disulfide bonds (II-V and IV-VI in HWTX-II, II-V and III-VI in the ICK molecules) is conserved both in HWTX-II and the ICK molecules, the structure of HWTX-II is unexpected absence of the cystine knot because of its unique disulfide linkage. It suggests that HWTX-II adopts a novel scaffold different from the ICK motif that is adopted by all other spider toxin structures elucidated thus far. Furthermore, the structure of HWTX-II, which conforms to the disulfide-directed beta-hairpin (DDH) motif, not only supports the hypothesis that the ICK is a minor elaboration of the more ancestral DDH motif but also suggests that HWTX-II may have evolved from the same structural ancestor.     

Solution structure of the carboxyl-terminal cysteine-rich domain of the VHv1.1 polydnaviral gene product: comparison with other cystine knot structural folds.

Polydnaviruses are an unusual group of insect viruses that have an obligate symbiotic association with certain parasitic wasps. These viruses are transmitted with the wasp egg during oviposition into lepidopteran insects, enabling the survival and development of the egg inside the host larvae. We report the three-dimensional structure of a novel polydnaviral cysteine-rich motif (cys-motif), identified as the carboxyl-terminal domain of a two cys-motif containing polydnaviral VHv1.1 gene product, abbreviated "C-term VHv1.1". This 65-residue domain was identified experimentally by limited proteolysis of the full-length protein and was subsequently cloned in a bacterial expression system for NMR studies. The C-term VHv1.1 3D structure was determined in solution by two-dimensional (1)H NMR spectroscopy. Calculation of the structure was based on a total of 300 upper distance restraints and 20 dihedral angle constraints, and resulted in an ensemble of 25 representative conformers with an average rmsd of 0.47 A from the mean structure for core backbone atoms. The protein core is made of a four beta-strand scaffold held together in a compact structure by three disulfide bonds, which form a cystine knot. The four beta-strands are arranged in an unusual configuration to form a triple-stranded beta-sheet and double-stranded beta-sheet. Comparison with other classes of cystine knots provides indication that C-term VHv1.1 represents a new and distinct cystine knot motif. This analysis provides a structural basis for interpretation of the genetic and amino acid sequence data classifying polydnavirus gene products as members of cysteine-rich protein families.     

The design of idealized alpha/beta-barrels: analysis of beta-sheet closure requirements

The 8-fold parallel alpha/beta-barrel topology is encountered in proteins that display an impressive variety of functions, suggesting that this topology may be a rather nonspecific and stable folding motif. Consequently, this motif can be considered as an interesting framework to design novel proteins. It has been shown that the shape of the beta-sheet portion of the barrel can be approximated by a hyperboloid. This geometric object may therefore be used as a scaffold to construct an idealized eight-stranded beta-barrel. To facilitate the de novo design of such structures, a collection of modeling tools has been developed allowing secondary structure elements to be mapped onto the scaffold surface and rotation and translation operations to be performed about user defined axes while evaluating their contribution to the conformational energy of the system. These tools have been applied in a systematic study assessing the phi, psi requirements to design symmetric eight stranded beta barrels with optimal hydrogen bonding between adjacent beta-strands. It is observed that: (a) the beta-sheet structure can be closed without introducing irregular stagger between beta-strands and (b) the region of phi, psi dihedral angle space compatible with the formation of regular symmetric eight stranded beta-barrels coincides with the phi, psi region corresponding to average beta-strands in known protein structures, suggesting that barrel closure does not impose gross constraints on beta-strand geometry.     

Importance of secondary structural specificity determinants in protein folding: insertion of a native beta-sheet sequence into an alpha-helical coiled-coil

To examine how a short secondary structural element derived from a native protein folds when in a different protein environment, we inserted an 11-residue beta-sheet segment (cassette) from human immunoglobulin fold, Fab new, into an alpha-helical coiled-coil host protein (cassette holder). This de novo design protein model, the structural cassette mutagenesis (SCM) model, allows us to study protein folding principles involving both short- and long-range interactions that affect secondary structure stability and conformation. In this study, we address whether the insertion of this beta-sheet cassette into the alpha-helical coiled-coil protein would result in conformational change nucleated by the long-range tertiary stabilization of the coiled-coil, therefore overriding the local propensity of the cassette to form beta-sheet, observed in its native immunoglobulin fold. The results showed that not only did the nucleating helices of the coiled-coil on either end of the cassette fail to nucleate the beta-sheet cassette to fold with an alpha-helical conformation, but also the entire chimeric protein became a random coil. We identified two determinants in this cassette that prevented coiled-coil formation: (1) a tandem dipeptide NN motif at the N-terminal of the beta-sheet cassette, and (2) the hydrophilic Ser residue, which would be buried in the hydrophobic core if the coiled-coil structure were to fold. By amino acid substitution of these helix disruptive residues, that is, either the replacement of the NN motif with high helical propensity Ala residues or the substitution of Ser with Leu to enhance hydrophobicity, we were able to convert the random coil chimeric protein into a fully folded alpha-helical coiled-coil. We hypothesized that this NN motif is a "secondary structural specificity determinant" which is very selective for one type of secondary structure and may prevent neighboring residues from adopting an alternate protein fold. These sequences with secondary structural specificity determinants have very strong local propensity to fold into a specific secondary structure and may affect overall protein folding by acting as a folding initiation site.     

Heparin-binding growth-associated molecule contains two heparin-binding beta -sheet domains that are homologous to the thrombospondin type I repeat

Heparin-binding growth-associated molecule (HB-GAM) is an extracellular matrix-associated protein implicated in the development and plasticity of neuronal connections of brain. Binding to cell surface heparan sulfate is indispensable for the biological activity of HB-GAM. In the present paper we have studied the structure of recombinant HB-GAM using heteronuclear NMR. These studies show that HB-GAM contains two beta-sheet domains connected by a flexible linker. Both of these domains contain three antiparallel beta-strands. In addition to this domain structure, HB-GAM contains the N- and C-terminal lysine-rich sequences that lack a detectable structure and appear to form random coils. Studies using CD and NMR spectroscopy suggest that HB-GAM undergoes a conformational change upon binding to heparin, and that the binding occurs primarily to the beta-sheet domains of the protein. Search of sequence data bases shows that the beta-sheet domains of HB-GAM are homologous to the thrombospondin type I repeat (TSR). Sequence comparisions show that the beta-sheet structures found previously in midkine, a protein homologous with HB-GAM, also correspond to the TSR motif. We suggest that the TSR sequence motif found in various extracellular proteins defines a beta-sheet structure similar to that found in HB-GAM and midkine. In addition to the apparent structural similarity, a similarity in biological functions is suggested by the occurrence of the TSR sequence motif in a wide variety of proteins that mediate cell-to-extracellular matrix and cell-to-cell interactions, in which the TSR domain mediates specific cell surface binding.     

Thermotoga maritima IscU. Structural characterization and dynamics of a new class of metallochaperone

Members of the IscU family of proteins are among the most conserved of all protein groups, extending across all three kingdoms of life. IscU serves as a scaffold for the assembly of intermediate iron-sulfur cluster centers and further mediates delivery to apo protein targets. Several proteins that mediate delivery of single metal ions to apo targets (termed metallochaperones) have recently been characterized structurally. Each displays a ferredoxin-like betaalphabetabetaalphabeta motif as a structural core. Assembly and delivery of a polynuclear iron-sulfur cluster is, however, a more complex pathway and presumably would demand a distinctive protein mediator. Here, we demonstrate Thermotoga maritima IscU (Tm IscU) to display unique structural and motional characteristics that distinguish it from other members of this class of proteins. In particular, IscU adopts a mobile, physiologically relevant, molten globule-like state that is vastly different from the previously identified ferredoxin-like fold that has thus far been characterized for other metallochaperones. The secondary structural content of Tm IscU is consistent with previous circular dichroism measurements on apo and holo protein, consisting of six alpha-helices and three beta-strands, the latter forming an anti-parallel beta-sheet. Extensive dynamics studies are consistent with a protein that has reasonably well defined secondary structural elements, but with a tertiary structure that is fluxional among widely different conformational arrangements. Analogous conformational flexibility does not exist in other structurally characterized metallochaperones; however, such a dynamic molecule may account for the lack of long-range NOEs, and allow both for the flexibility that is necessary for the multiple roles of Fe-S cluster assembly, and recognition and delivery of that cluster to a target protein. Additionally, the fluxionality of IscU is unique in that the protein appears to be more compact (based on 1H/2H exchange, R1, R2, and NOE data) but yet more fluid (lack of long-range NOEs) than typical molten globule proteins.     

RNA recognition motifs: boring? Not quite

The RNA recognition motif (RRM) is one of the most abundant protein domains in eukaryotes. While the structure of this domain is well characterized by the packing of two alpha-helices on a four-stranded beta-sheet, the mode of protein and RNA recognition by RRMs is not clear owing to the high variability of these interactions. Here we report recent structural data on RRM-RNA and RRM-protein interactions showing the ability of this domain to modulate its binding affinity and specificity using each of its constitutive elements (beta-strands, loops, alpha-helices). The extreme structural versatility of the RRM interactions explains why RRM-containing proteins have so diverse biological functions.     

A common structural motif incorporating a cystine knot and a triple-stranded beta-sheet in toxic and inhibitory polypeptides.

A common structural motif consisting of a cystine knot and a small triple-stranded beta-sheet has been defined from comparison of the 3-dimensional structures of the polypeptides omega-conotoxin GVIA (Conus geographus), kalata BI (Oldenlandia affinis DC), and CMTI-I (Curcurbita maxima). These 3 polypeptides have diverse biological activities and negligible amino acid sequence identity, but each contains 3 disulfide bonds that give rise to a cystine knot. This knot consists of a ring formed by the first 2 bonds (1-4 and 2-5) and the intervening polypeptide backbone, through which the third disulfide (3-6) passes. The other component of this motif is a triple-stranded, anti-parallel beta-sheet containing a minimum of 10 residues, XXC2, XC5X, XXC6X (where the numbers on the half-cysteine residues refer to their positions in the disulfide pattern). The presence in these polypeptides of both the cysteine knot and antiparallel beta-sheet suggests that both structural features are required for the stability of the motif. This structural motif is also present in other protease inhibitors and a spider toxin. It appears to be one of the smallest stable globular domains found in proteins and is commonly used in toxins and inhibitors that act by blocking the function of larger protein receptors such as ion channels or proteases.     

Structural trees for proteins containing φ-motifs

In the present study, a novel structural motif of proteins referred to as the phi-motif is considered, and two novel structural trees in which the phi-motif is taken as the root structure have been constructed. The simplest phi-motif is formed by three adjacent beta-strands connected by loops and packed in one beta-sheet so that its overall fold resembles the Greek letter phi. Construction of the structural trees and modeling of folding pathways have shown that all structures of the protein superfamilies can be obtained by stepwise addition of alpha-helices and/or beta-strands to the root phi-motif taking into account a restricted set of rules inferred from known principles of protein structure. The structural trees are a good tool for structure comparison, structural classification of proteins, as well as for searching for all possible protein folds and folding pathways.     

Alanine substitutions of noncysteine residues in the cysteine‐stabilized αβ motif

The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine‐stabilized αβ motif (CSαβ) consists of an α‐helix and an antiparallel triple‐stranded β‐sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSαβ motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty‐two of 46 residue positions of VrD1 are altered by site‐directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering.     

The structure of the MAPK scaffold, MP1, bound to its partner, p14. A complex with a critical role in endosomal map kinase signaling

Scaffold proteins of the mitogen-activated protein kinase (MAPK) pathway have been proposed to form an active signaling module and enhance the specificity of the transduced signal. Here, we report a 2-A resolution structure of the MAPK scaffold protein MP1 in a complex with its partner protein, p14, that localizes the complex to late endosomes. The structures of these two proteins are remarkably similar, with a five-stranded beta-sheet flanked on either side by a total of three helices. The proteins form a heterodimer in solution and interact mainly through the edge beta-strand in each protein to generate a 10-stranded beta-sheet core. Both proteins also share structural similarity with the amino-terminal regulatory domains of the membrane trafficking proteins, sec22b and Ykt6p, as well as with sedlin (a component of a Golgi-associated membrane-trafficking complex) and the sigma2 and amino-terminal portion of the mu2 subunits of the clathrin adaptor complex AP2. Because neither MP1 nor p14 have been implicated in membrane traffic, we propose that the similar protein folds allow these relatively small proteins to be involved in multiple and simultaneous protein-protein interactions. Mapping of highly conserved, surface-exposed residues on MP1 and p14 provided insight into the potential sites of binding of the signaling kinases MEK1 and ERK1 to this complex, as well as the areas potentially involved in other protein-protein interactions.     

Identification of the RNA binding domain of T4 RegA protein by structure-based mutagenesis.

The T4 translational repressor RegA protein folds into two structural domains, as revealed by the crystal structure (Kang, C.-H. , Chan, R., Berger, I., Lockshin, C., Green, L., Gold, L., and Rich, A. (1995) Science 268, 1170-1173). Domain I of the RegA protein contains a four-stranded beta-sheet and two alpha-helices. Domain II contains a four-stranded beta-sheet and an unusual 3/10 helix. Since beta-sheet residues play a role in a number of protein-RNA interactions, one or both of the beta-sheet regions in RegA protein may be involved in RNA binding. To test this possibility, mutagenesis of residues on both beta-sheets was performed, and the effects on the RNA binding affinities of RegA protein were measured. Additional sites for mutagenesis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluorescence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shift assays using lysed cell supernatants. The results of this mutagenesis study ruled out the participation of beta-sheet residues. Instead, the RNA binding site was found to be a surface pocket formed by residues on two loops and an alpha-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition properties.     

In Search of Functional Advantages of Knots in Proteins

We analysed the structure of deeply knotted proteins representing three unrelated families of knotted proteins. We looked at the correlation between positions of knotted cores in these proteins and such local structural characteristics as the number of intra-chain contacts, structural stability and solvent accessibility. We observed that the knotted cores and especially their borders showed strong enrichment in the number of contacts. These regions showed also increased thermal stability, whereas their solvent accessibility was decreased. Interestingly, the active sites within these knotted proteins preferentially located in the regions with increased number of contacts that also have increased thermal stability and decreased solvent accessibility. Our results suggest that knotting of polypeptide chains provides a favourable environment for the active sites observed in knotted proteins. Some knotted proteins have homologues without a knot. Interestingly, these unknotted homologues form local entanglements that retain structural characteristics of the knotted cores.     

Cross-strand disulphides in cell entry proteins: poised to act

Cross-strand disulphides (CSDs) are unusual bonds that link adjacent strands in the same beta-sheet. Their peculiarity relates to the high potential energy stored in these bonds, both as torsional energy in the highly strained disulphide linkage and as deformation energy stored in the sheet itself. CSDs are relatively rare in protein structures but are conspicuous by their presence in proteins that are involved in cell entry. The finding that entry of botulinum neurotoxin and HIV into mammalian cells involves cleavage of CSDs suggests that the activity of other cell entry proteins may likewise involve cleavage of these bonds. We examine emerging evidence of the involvement of these unusual disulphides in cell entry events.