NMR structure and regulated expression in APL cell of human SH3BGRL3

SH3 domain binding glutamic acid-rich protein like 3 (SH3BGRL3) is the new member of thioredoxin (TRX) super family, whose posttranslational modified form was identified as tumor necrosis factor alpha (TNF-alpha) inhibitory protein, TIP-B1. In this paper, we determined its solution structure by multi-dimensional nuclear magnetic resonance spectroscopy. The overall structure of human SH3BGRL3 conformed to a TRX-like fold. To understand its function in vivo, the upregulated expression in acute promyelocytic leukemia cell line NB4 at both mRNA and protein level was elucidated. Immunofluorescence and immunohistochemistry staining with monoclonal antibody against SH3BGRL3 demonstrated that it was a cytoplasmic protein in both NB4 cell and human tissues. These results, as a whole, indicate that SH3BGRL3 may function as a regulator in all-trans retinoic acid-induced pathway.     

The crystal structure of the olfactory marker protein at 2.3 A resolution

Olfactory marker protein (OMP) is a highly expressed and phylogenetically conserved cytoplasmic protein of unknown function found almost exclusively in mature olfactory sensory neurons. Electrophysiological studies of olfactory epithelia in OMP knock-out mice show strongly retarded recovery following odorant stimulation leading to an impaired response to pulsed odor stimulation. Although these studies show that OMP is a modulator of the olfactory signal-transduction cascade, its biochemical role is not established. In order to facilitate further studies on the molecular function of OMP, its crystal structure has been determined at 2.3 A resolution using multiwavelength anomalous diffraction experiments on selenium-labeled protein. OMP is observed to form a modified beta-clamshell structure with eight antiparallel beta-strands. While OMP has no significant sequence homology to proteins of known structure, it has a similar fold to a domain found in a variety of existing structures, including in a large family of viral capsid proteins. The surface of OMP is mostly convex and lacking obvious small molecule binding sites, suggesting that it is more likely to be involved in modulating protein-protein interaction than in interacting with small molecule ligands. Three highly conserved regions have been identified as leading candidates for protein-protein interaction sites in OMP. One of these sites represents a loop known to mediate ligand interactions in the structurally homologous EphB2 receptor ligand-binding domain. This site is partially buried in the crystal structure but fully exposed in the NMR solution structure of OMP due to a change in the orientation of an alpha-helix that projects outward from the structurally invariant beta-clamshell core. Gating of this conformational change by molecular interactions in the signal-transduction cascade could be used to control access to OMP's equivalent of the EphB2 ligand-interaction loop, thereby allowing OMP to function as a molecular switch.     

The high resolution NMR structure of the third SH3 domain of CD2AP

CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney function. CD2AP interacts, as an adaptor protein, with different natural targets, such as CD2, nefrin, c-Cbl and podocin. These proteins are believed to interact to one of the three SH3 domains that are positioned in the N-terminal region of CD2AP. To understand the network of interactions between the natural targets and the three SH3 domains (SH3-A, B and C), we have started to determine the structures of the individual SH3 domains. Here we present the high-resolution structure of the SH3-C domain derived from NMR data. Full backbone and side-chain assignments were obtained from triple-resonance spectra. The structure was determined from distance restraints derived from high-resolution 600 and 800 MHz NOESY spectra, together with phi and psi torsion angle restraints based on the analysis of ¹HN, ¹⁵N, ¹Hα, ¹³Cα, ¹³CO and ¹³Cβ chemical shifts. Structures were calculated using CYANA and refined in water using RECOORD. The three-dimensional structure of CD2AP SH3-C contains all the features that are typically found in other SH3 domains, including the general binding site for the recognition of polyproline sequences.     

Crystal structure of alpha-hordothionin at 1.9 Angstrom resolution

Crystal structure of ubiquitous toxin from barley alpha-hordothionin (alpha-HT) has been determined at 1.9A resolution by X-ray crystallography. The primary sequence as well as the NMR solution structure of alpha-HT firmly established that alpha-HT belongs to a family of membrane active plant toxins-thionins. Since alpha-HT crystallized in a space group (P4(1)2(1)2) that is different from the space group (I422) of previously determined alpha(1)- and beta-purothionins, and visocotoxin A3, therefore, it provided independent information on protein-protein interactions that may be relevant to the toxin mechanism. The structure of alpha-HT not only confirms overall architectural features (crambin fold) but also provides an additional confirmation of the role for crucial solute molecules, that were postulated to be directly involved in the mechanism of toxicity for thionins.     

Implications of SH3 domain structure and dynamics for protein regulation and drug design

SH3 Domains provide interesting targets for investigations of protein structure and dynamics because of their compact size and importance for signal transduction. The present review summarizes recent research investigating SH3 domain structure and dynamics, the discovery of novel SH3 domains, the role of SH3 domains in disease, and progress in targeting SH3 domains for the development of novel therapeutics. Particular emphasis is placed on the unfolding/refolding characteristics of SH3 domains and the potential importance of these processes for regulation of signal transduction.     

Crystal Structure of the Src Family Kinase Hck SH3-SH2 Linker Regulatory Region Supports an SH3-dominant Activation Mechanism

Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity. However, the amino acid sequence of the Hck linker represents a suboptimal ligand for the isolated SH3 domain, suggesting that it may form the polyproline type II helical conformation required for SH3 docking only in the context of the intact structure. To test this hypothesis directly, we determined the crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker. Despite the absence of the kinase domain, the structures and relative orientations of the SH2 and SH3 domains in this shorter protein were very similar to those observed in near full-length, down-regulated Hck. However, the SH2 kinase linker adopted a modified topology and failed to engage the SH3 domain. This new structure supports the idea that these noncatalytic regions work together as a “conformational switch” that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hck protein. Our results also provide fresh structural insight into the facile induction of Hck activity by HIV-1 Nef and other Hck SH3 domain binding proteins and implicate the existence of innate conformational states unique to individual Src family members that “fine-tune” their sensitivities to activation by SH3-based ligands.     

Crystal structure of a conger eel galectin (congerin II) at 1.45A resolution: implication for the accelerated evolution of a new ligand-binding site following gene duplication

The crystal structure of congerin II, a galectin family lectin from conger eel, was determined at 1.45A resolution. The previously determined structure of its isoform, congerin I, had revealed a fold evolution via strand swap; however, the structure of congerin II described here resembles other prototype galectins. A comparison of the two congerin genes with that of several other galectins suggests acceralated evolution of both congerin genes following gene duplication. The presence of a Mes (2-[N-morpholino]ethanesulfonic acid) molecule near the carbohydrate-binding site in the crystal structure points to the possibility of an additional binding site in congerin II. The binding site consists of a group of residues that had been replaced following gene duplication suggesting that the binding site was built under selective pressure. Congerin II may be a protein specialized for biological defense with an affinity for target carbohydrates on parasites' cell surface.     

High-Resolution Crystal Structure of Spectrin SH3 Domain Fused with a Proline-Rich Peptide

Abstract

A new chimeric protein, named WT-CIIA, was designed by connecting the proline-rich decapeptide PPPVPPYSAG to the C-terminus of the alpha-spectrin SH3 domain through a natural twelve-residue linker to obtain a single-chain model that would imitate intramolecular SH3-ligand interaction. The crystal structure of this fusion protein was determined at 1.7 Å resolution. The asymmetric unit of the crystal contained two SH3 globules contacting with one PPPVPPY fragment located between them. The domains are related by the twofold non-crystallographic axis and the ligand lies in two opposite orientations with respect to the conservative binding sites of SH3 domains.     

The structure of the plakin domain of plectin reveals a non-canonical SH3 domain interacting with its fourth spectrin repeat

Plectin belongs to the plakin family of cytoskeletal crosslinkers, which is part of the spectrin superfamily. Plakins contain an N-terminal conserved region, the plakin domain, which is formed by an array of spectrin repeats (SR) and a Src-homology 3 (SH3), and harbors binding sites for junctional proteins. We have combined x-ray crystallography and small angle x-ray scattering (SAXS) to elucidate the structure of the central region of the plakin domain of plectin, which corresponds to the SR3, SR4, SR5, and SH3 domains. The crystal structures of the SR3-SR4 and SR4-SR5-SH3 fragments were determined to 2.2 and 2.95 Å resolution, respectively. The SH3 of plectin presents major alterations as compared with canonical Pro-rich binding SH3 domains, suggesting that plectin does not recognize Pro-rich motifs. In addition, the SH3 binding site is partially occluded by an intramolecular contact with the SR4. Residues of this pseudo-binding site and the SR4/SH3 interface are conserved within the plakin family, suggesting that the structure of this part of the plectin molecule is similar to that of other plakins. We have created a model for the SR3-SR4-SR5-SH3 region, which agrees well with SAXS data in solution. The three SRs form a semi-flexible rod that is not altered by the presence of the SH3 domain, and it is similar to those found in spectrins. The flexibility of the plakin domain, in analogy with spectrins, might contribute to the role of plakins in maintaining the stability of tissues subject to mechanical stress.     

Determination of the solution structure of the SH3 domain of human p56 Lck tyrosine kinase

Summary The solution structure of the SH3 domain of human p56 Lck tyrosine kinase (Lck-SH3) has been determined by multidimensional heteronuclear NMR spectroscopy. The structure was calculated from a total of 935 experimental restraints comprising 785 distance restraints derived from 1017 assigned NOE cross peaks and 150 dihedral angle restraints derived from 160 vicinal coupling constants. A novel combination of the constant-time ¹H–¹³C NMR correlation experiment recorded with various delays of the constant-time refocusing delays and a fractionally ¹³C-labelled sample was exploited for the stereo-specific assignment of prochiral methyl groups. Additionally, 28 restraints of 14 identified hydrogen bonds were included. A family of 25 conformers was selected to characterize the solution structure. The average root-mean-square deviations of the backbone atoms (N, C, C, O) among the 25 conformers is 0.42 Å for residues 7 to 63. The N- and C-terminal residues, 1 to 6 and 64 to 81, are disordered, while the well-converged residues 7 to 63 correspond to the conserved sequences of other SH3 domains. The topology of the SH3 structure comprises five anti-parallel -strands arranged to form two perpendicular -sheets, which are concave and twisted in the middle part. The overall secondary structure and the backbone conformation of the core -strands are almost identical to the X-ray structure of the fragment containing the SH2-SH3 domains of p56 Lck [Eck et al. (1994) Nature, 368, 764–769]. The X-ray structure of the SH3 domain in the tandem SH2-SH3 fragment is spatially included within the ensemble of the 25 NMR conformers, except for the segment of residues 14 to 18, which makes intermolecular contacts with an adjacent SH2 molecule and the phosphopeptide ligand in the crystal lattice. Local structural differences from other known SH3 domains are also observed, the most prominent of which is the absence in Lck-SH3 of the two additional short -strands in the regions Ser¹⁵ to Glu¹⁷ and Gly²⁵ to Glu²⁷ flanking the so-called RT-Src loop. This loop (residues Glu¹⁷ to Leu²⁴), together with the n-Src loop (residues Gln³⁷ to Ser⁴⁶) confines the ligand interaction site which is formed by a shallow patch of hydrophobic amino acids (His¹⁴, Tyr¹⁶, Trp⁴¹, Phe⁵⁴ and Phe⁵⁹). Both loops are flexible and belong to the most mobile regions of the protein, which is assessed by the heteronuclear ¹⁵N,¹H-NOE values characterizing the degree of internal backbone motions. The aromatic residues of the ligand binding site are arranged such that they form three pockets for interactions with the polyproline ligand.Abbreviations CT constant time - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - SH2 Src homology domain 2 - SH3 Src homology domain 3     

Folding of the alphaII-spectrin SH3 domain under physiological salt conditions

The SH3 domain has often been used as a model for protein folding due to its typical two-state behaviour. However, recent experimental data at low pH as well as molecular dynamic simulations have indicated that the folding process of SH3 probably is more complicated, and may involve intermediate states. Using both kinetic and equilibrium measurements we have obtained evidence that under native-like conditions the folding of the spectrin SH3 domain does not follow a classic two-state behaviour. The curvature we observed in the Chevron plots is a strong indication of a non-linear activation energy relationship due to the presence of high-energy intermediates. In addition, circular dichroism measurements indicated that refolding after thermal denaturation did not follow the same pattern as thermal unfolding but rather implied less cooperativity and that the refolding transition increased with increasing protein concentration. Further, NMR experiments indicated that upon refolding the SH3 domain gave rise to more than one conformation. Therefore, our results suggest that the folding of the SH3 domain of αII-spectrin does not follow a classical two-state process under high-salt conditions and neutral pH. Heterogeneous folding pathways, which can include folding intermediates as well as misfolded intermediates, might give a more reasonable insight into the folding behaviour of the αII-spectrin SH3 domain.     

Crystal structure of human immunoglobulin fragment Fab New refined at 2.0 A resolution.

The three-dimensional structure of the human immunoglobulin fragment Fab New (IgG1, lambda) has been refined to a crystallographic R-factor of 16.9% to 2 A resolution. Rms deviations of the final model from ideal geometry are 0.014 A for bond distances and 3.03 degrees for bond angles. Refinement was based on a new X-ray data set including 28,301 reflections with F > 2.5 sigma(F) from 6.0 to 2.0 A resolution. The starting model for the refinement procedure reported here is from the Brookhaven Protein Data Bank entry 3FAB (rev. 1981). Differences between the initial and final models include modified polypeptide-chain folding in the third complementarity-determining region (CDR3) and the third framework region (FR3) of VH and in some exposed loops of CL and CH1. Amino acid sequence changes were determined at a number of positions by inspection of difference electron density maps. The incorporation of amino acid sequence changes results in an improved VH framework model for the "humanization" of monoclonal antibodies.     

Crystal structure of Ca2+ -free S100A2 at 1.6-A resolution

S100A2 is an EF hand-containing Ca^2 +-binding protein of the family of S100 proteins. The protein is localized exclusively in the nucleus and is involved in cell cycle regulation. It attracted most interest by its function as a tumor suppressor via p53 interaction. We determined the crystal structure of homodimeric S100A2 in the Ca^2 +-free state at 1.6-Å resolution. The structure revealed structural differences between subunits A and B, especially in the conformation of a loop that connects the N- and C-terminal EF hands and represents a part of the target-binding site in S100 proteins. Analysis of the hydrogen bonding network and molecular dynamics calculations indicate that one of the two observed conformations is more stable. The structure revealed Na⁺ bound to each N-terminal EF hand of both subunits coordinated by oxygen atoms of the backbone carbonyl and water molecules. Comparison with the structures of Ca^2 +-free S100A3 and S100A6 suggests that Na⁺ might occupy the S100-specific EF hand in the Ca^2 +-free state.     

Exon Inclusion Modulates Conformational Plasticity and Autoinhibition of the Intersectin 1 SH3A Domain

1. Download : Download high-res image (223KB)
2. Download : Download full-size image

     

Crystal structure of the N-terminal SH3 domain of mouse betaPIX, p21-activated kinase-interacting exchange factor

The mouse betaPIX-SH3 domain, residues 8-63 of P21-activated kinase interacting exchange factor, has been characterized by X-ray diffraction. Crystals belonging to space group P3(2)21 diffracted to 2.0 A and the structure was phased by the single-wavelength anomalous diffraction method. The domain is a compact beta-barrel with an overall conformation similar to the general SH3 structure. The X-ray structure shows mouse betaPIX-SH3 domain binding the way in which the betaPIX characteristic amino acids do so for an unconventional ligand binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by mouse betaPIX-SH3 domain. Comparison with another SH3/peptide complex shows that the recognition mode of the mouse betaPIX-SH3 domain should be very similar to the RXXK ligand binding mode. The unique large and planar hydrophobic pocket may contribute to the promiscuity of betaPIX-SH3 domain resulting in its multiple biological functions.     

Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags

Small peptide tags are often fused to proteins to allow their affinity purification in high-throughput structure analysis schemes. To assess the compatibility of small peptide tags with protein crystallization and to examine if the tags alter the three-dimensional structure, the N-terminus of the chicken alpha-spectrin SH3 domain was labeled with a His6 tag and the C-terminus with a StrepII tag. The resulting protein, His6-SH3-StrepII, consists of 83 amino-acid residues, 23 of which originate from the tags. His6-SH3-StrepII is readily purified by dual affinity chromatography, has very similar biophysical characteristics as the untagged protein domain and crystallizes readily from a number of sparse-matrix screen conditions. The crystal structure analysis at 2.3 A resolution proves native-like structure of His6-SH3-StrepII and shows the entire His6 tag and part of the StrepII tag to be disordered in the crystal. Obviously, the fused affinity tags did not interfere with crystallization and structure analysis and did not change the protein structure. From the extreme case of His6-SH3-StrepII, where affinity tags represent 27% of the total fusion protein mass, we extrapolate that protein constructs with N- and C-terminal peptide tags may lend themselves to biophysical and structural investigations in high-throughput regimes.     

The 1.1 A resolution crystal structure of the p130cas SH3 domain and ramifications for ligand selectivity

The Crk-associated tyrosine kinase substrate p130cas (CAS) is a docking protein containing an SH3 domain near its N terminus, followed by a short proline-rich segment, a large central substrate domain composed of 15 repeats of the four amino acid sequence YxxP, a serine-rich region and a carboxy-terminal domain, which possesses consensus binding sites for the SH2 and SH3 domains of Src (YDYV and RPLPSPP, respectively). The SH3 domain of CAS mediates its interaction with several proteins involved in signaling pathways such as focal adhesion kinase (FAK), tyrosine phosphatases PTP1B and PTP-PEST, and the guanine nucleotide exchange factor C3G. As a homolog of the corresponding Src docking domain, the CAS SH3 domain binds to proline-rich sequences (PxxP) of its interacting partners that can adopt a polyproline type II helix. We have determined a high-resolution X-ray structure of the recombinant human CAS SH3 domain. The domain, residues 1-69, crystallized in two related space groups, P2(1) and C222(1), that provided diffraction data to 1.1 A and 2.1 A, respectively. The crystal structure shows, in addition to the conserved SH3 domain architecture, the way in which the CAS characteristic amino acids form an atypically charged ligand-binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by the CAS SH3 domain. The structure enables modelling of the docking interactions to its ligands, for example from focal adhesion kinase, and supports structure-based drug design of inhibitors of the CAS-FAK interaction.     

Design and structural thermodynamic studies of the chimeric protein derived from spectrin SH3 domain

A number of the chimeric constructs with spectrin SH3 domain were designed for structural and thermodynamic studies of protein self-assembly and protein-ligand interactions. SH3 domains, components of many regulatory proteins, operate through weak interactions with proline-rich regions of polypeptide chains. The recombinant construct (WT-CIIA) studied in this work was constructed by linking the peptide ligand PPPVPPYSAG to the domain C-terminus via a long 12-residue linker to increase the affinity of this ligand for the spectrin domain, thereby ensuring a stable positioning of the polyproline helix to the conserved ligand-binding site in orientation II, which is regarded as untypical of the interaction between this domain and oligopeptides. A comparison of fluorescence spectra of the initial domain and the recombinant protein WT-CIIA suggests that the ligand sticks to the conservative binding site. However, analysis of the equilibrium urea-induced unfolding has demonstrated that this is an unstable contact, which leads to a two-stage unfolding of the chimeric protein. The protein WT-CIIA was crystallized; a set of X-ray diffraction data with a resolution of 1.75 Å was recorded from individual crystals. A preliminary analysis of these diffraction data has demonstrated that the crystals belong to space group P32 with the following unit cell parameters: a = b = 36.39, c = 112.17 Å, a = β = 90.0, and γ = 120.0.     

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.     

Structural and thermodynamic basis for the interaction of the Src SH2 domain with the activated form of the PDGF beta-receptor

Recruitment of the Src kinase to the activated form of the platelet-derived growth factor (PDGF) receptor involves recognition of a unique sequence motif in the juxtamembrane region of the receptor by the Src homology 2 (SH2) domain of the enzyme. This motif contains two phosphotyrosine residues separated by one residue (sequence pYIpYV where pY indicates a phosphotyrosine). Here, we provide the thermodynamic and structural basis for the binding of this motif by the Src SH2 domain. We show that the second phosphorylation event increases the free energy window for specific interaction and that the physiological target is exquisitely designed for the task of recruiting specifically an SH2 domain which otherwise demonstrates very little intrinsic ability to discriminate sequences C-terminal to the first phosphorylation event. Surprisingly, we show that water plays a role in the recognition process.