Src homology 2 domain-containing protein tyrosine phosphatase [SHP] substrate 1 (SHPS-1), a receptor-type transmembrane glycoprotein whose cytoplasmic region binds and activates the protein tyrosine phosphatases SHP-1 and SHP-2, and thereby modulates multiple cellular functions.Its extracellular region regulates intercellular communication in the neural and immune systems through its association with CD47 on adjacent cells.Interactions between CD47 and SHPS-1 are implicated in multiple cellular processes, including cell motility [1], neutrophil transmigration, phagocytosis of red blood cells by splenic macrophages [2], and T cell activation.Although the roles of the CD47-SHPS-1 system has been presented, little is known about the cell surface organization of these ligand/receptor complexes and the structural basis for signal transduction.To gain new insights into the physiological and biological roles of the CD47-SHPS-1 system, we determined the crystal structure of the SHPS-1 extracellular domain.The domain adopts a classical immunoglobulin (Ig) fold that was observed to form an antiparallel dimer.A dimeric form of SHPS-1 was observed in vivo, and our structural and biophysical data shows that the extracellular domain of SHPS-1 is dimeric in solution, compatible with the view of SHPS-1 acting as a cis-dimeric adhesion receptor.Previous investigations showed native CD47 formed cis-dimers.These features suggest that both CD47 and SHPS-1 trans-interact each other by the formation of cis-dimers and offer perceptions into interactions of related Ig superfamily receptors.

The crystal structure of a complex of methyl-alpha-D-mannoside with banana lectin from Musa paradisiaca reveals two primary binding sites in the lectin, unlike in other lectins with beta-prism I fold which essentially consists of three Greek key motifs. It has been suggested that the fold evolved through successive gene duplication and fusion of an ancestral Greek key motif. In other lectins, all from dicots, the primary binding site exists on one of the three motifs in the three-fold symmetric molecule. Banana is a monocot, and the three motifs have not diverged enough to obliterate sequence similarity among them. Two Greek key motifs in it carry one primary binding site each. A common secondary binding site exists on the third Greek key. Modelling shows that both the primary sites can support 1-2, 1-3, and 1-6 linked mannosides with the second residue interacting in each case primarily with the secondary binding site. Modelling also readily leads to a bound branched mannopentose with the nonreducing ends of the two branches anchored at the two primary binding sites, providing a structural explanation for the lectin's specificity for branched alpha-mannans. A comparison of the dimeric banana lectin with other beta-prism I fold lectins, provides interesting insights into the variability in their quaternary structure.

The structure of Mycobacterium smegmatis single-stranded DNA-binding protein (SSB) has been determined using three data sets collected from related crystals. The structure is similar to that of its homologue from Mycobacterium tuberculosis, indicating that the clamp arrangement that stabilizes the dimer and the ellipsoidal shape of the tetramer are characteristic features of mycobacterial SSBs. The central OB fold is conserved in mycobacterial SSBs as well as those from Escherichia coli, Deinococcus radiodurans and human mitochondria. However, the quaternary structure exhibits considerable variability. The observed plasticity of the subunit is related to this variability. The crystal structures and modelling provide a rationale for the variability. The strand involved in the clamp mechanism, which leads to higher stability of the tetramer, appears to occur in all high-G+C Gram-positive bacteria. The higher stability is perhaps required by these organisms. The mode of DNA binding of mycobacterial SSBs is different from that of E. coli SSB partly on account of the difference in the shape of the tetramers. Another difference between the two modes is that the former contains additional ionic interactions and is more susceptible to salt concentration.

The crystal structure of Mycobacterium tuberculosis ribosome recycling factor has been determined and refined against three X-ray diffraction data sets, two collected at room temperature and the other at 100 K. The two room-temperature data sets differ in the radiation damage suffered by the crystals before the data used for processing were collected. A comparison between the structures refined against the two data sets indicates the possibility of radiation-induced conformational change. The L-shaped molecule is composed of a long three-helix bundle domain (domain I) and a globular domain (domain II) connected by a linker region. The main difference between the room-temperature structure and the low temperature structure is in the rotation of domain II about an axis close to its libration axis. This observation and a detailed comparative study of ribosome recycling factors (RRFs) of known structures led to an elaboration of the present understanding of the structural variability of RRF. The variability involves a change in the angle between the two arms of the molecule, a rotation of domain II in a plane nearly perpendicular to the axis of the helix bundle and an internal rotation of domain II. Furthermore, the domains and the linker could be delineated into fixed and variable regions in a physically meaningful manner. The relative mobility of the domains of the molecule in the crystal structure appears to be similar to that in the ribosome–RRF complex. That permits a meaningful discussion of the structural features of RRF in terms of ribosome–RRF interactions. The structure also provides insights into the results of inter-species complementation studies.

The ribosome recycling factor from Mycobacterium tuberculosis has been crystallized. The monoclinic crystals, with 52.5% solvent content, contain one protein molecule in the asymmetric unit.

The ribosome recycling factor from Mycobacterium tuberculosis has been crystallized. The monoclinic crystals, with 52.5% solvent content, contain one protein molecule in the asymmetric unit.

The banana lectin from Musa paradisiaca, MW 29.4 kDa, has been isolated, purified and crystallized. The trigonal crystals contain one dimeric molecule in the asymmetric unit. The structure has been solved using molecular replacement to a resolution of 3 A. The structure of the subunit is similar to that of jacalin-like lectins.

Single-stranded DNA-binding protein (SSB) is an essential protein necessary for the functioning of the DNA replication, repair and recombination machineries. Here we report the structure of the DNA-binding domain of Mycobacterium tuberculosis SSB (MtuSSB) in four different crystals distributed in two forms. The structure of one of the forms was solved by a combination of isomorphous replacement and anomalous scattering. This structure was used to determine the structure of the other form by molecular replacement. The polypeptide chain in the structure exhibits the oligonucleotide binding fold. The globular core of the molecule in different subunits in the two forms and those in Escherichia coli SSB (EcoSSB) and human mitochondrial SSB (HMtSSB) have similar structure, although the three loops exhibit considerable structural variation. However, the tetrameric MtuSSB has an as yet unobserved quaternary association. This quaternary structure with a unique dimeric interface lends the oligomeric protein greater stability, which may be of significance to the functioning of the protein under conditions of stress. Also, as a result of the variation in the quaternary structure the path adopted by the DNA to wrap around MtuSSB is expected to be different from that of EcoSSB.

Uracil DNA glycosylase (UDG), a ubiquitous and highly specific enzyme, commences the uracil excision repair pathway. Structural studies have shown that the tyrosine in a highly conserved GQDPY water-activating loop of UDGs blocks the entry of thymine or purines into the active site pocket. To further understand the role of this tyrosine (Y66 in Escherichia coli UDG), we have overproduced and characterized Y66F, Y66H, Y66L and Y66W mutants. The complexes of the wild-type, Y66F, Y66H and Y66L UDGs with uracil DNA glycosylase inhibitor (Ugi) (a proteinaceous substrate mimic) were stable to 8 M urea. However, some dissociation of the complex involving the Y66W UDG occurred at this concentration of urea. The catalytic efficiencies (V(max) / K(m)) of the Y66L and Y66F mutants were similar to those of the wild-type UDG. However, the Y66W and Y66H mutants were approximately 7- and approximately 173-fold compromised, respectively, in their activities. Interestingly, the Y66W mutation has resulted in an enzyme which is resistant to product inhibition. Preferential utilization of a substrate enabling a long range contact between the -5 phosphate (upstream to the scissile uracil) and the enzyme, and the results of modeling studies showing that the uracil-binding cavity of Y66W is wider than those of the wild type and other mutant UDGs, suggest a weaker interaction between uracil and the Y66W mutant. Furthermore, the fluorescence spectroscopy of UDGs and their complexes with Ugi, in the presence of uracil or its analog, 5-bromouracil, suggests compromised binding of uracil in the active site pocket of the Y66W mutant. Lack of inhibition of the Y66W UDG by apyrimidinic DNA (AP-DNA) is discussed to highlight a potential additional role of Y66 in shielding the toxic effects of AP-DNA, by lowering the rate of its release for subsequent recognition by an AP endonuclease.

The structures of a new crystal form of free Escherichia coli uracil DNA glycosylase (UDG), containing four molecules in the asymmetric unit, and two forms of its complex with the proteinaceous inhibitor Ugi, containing two and four crystallographically independent complexes, have been determined. A comparison of these structures and the already known crystal structures containing UDG shows that the enzyme can be considered to be made up of two independently moving structural entities or domains. A detailed study of free and DNA-bound human enzyme strengthens this conclusion. The domains close upon binding to uracil-containing DNA, whereas they do not appear to do so upon binding to Ugi. The comparative study also shows that the mobility of the molecule involves the rigid-body movement of the domains superposed on flexibility within domains.

NCI at Frederick (bZIP) DNA-binding proteins play key roles in regulating gene transcription.Their DNA binding domains consist of the basic region juxtaposed to a heptad repeats of leucines.bZIP proteins from the same family recognize a unique palindromic DNA motif.bZIP peptide binds to DNA as a dimer of α helices.Dimerization is mediated by the leucine zipper; each basic region contacts a half-site in the major groove of the DNA.We solved the crystal structure of the C/EBPα bZIP peptide bound to a 21-mer DNA duplex.The current R-factor is 22.42% (R-free is 28.06%) in the 40 to 2.8 Å resolution range.Residues conserved among bZIP proteins -Asn292, Ala295 and Arg300 -make DNA contacts that are analogous to those observed in GCN4, CREB and Fos/Jun.Critical for the specific DNA binding by C/EBP proteins is the conformation of Arg289, which makes electrostatic interactions with Asn292, Asn293, and the adenine at position 3 (A3) in the consensus half-site (GCAAT).Val296 makes van der Waals contact with the methyl group of thymine which forms a base pair with A3.We also show that mutation of Arg289 to Ala completely abolishes the C/EBP α DNA binding, whereas a mutant in which Val296 is changed to Ala binds with comparable affinity to the C/EBP and CREB (halfsite = GTCAT) sites.