Fibroblast growth factors 1 and 2 are distinct in oligomerization in the presence of heparin-like glycosaminoglycans.
(33/24796)
Fibroblast growth factor (FGF) 1 and FGF-2 are prototypic members of the FGF family, which to date comprises at least 18 members. Surprisingly, even though FGF-1 and FGF-2 share more than 80% sequence similarity and an identical structural fold, these two growth factors are biologically very different. FGF-1 and FGF-2 differ in their ability to bind isoforms of the FGF receptor family as well as the heparin-like glycosaminoglycan (HLGAG) component of proteoglycans on the cell surface to initiate signaling in different cell types. Herein, we provide evidence for one mechanism by which these two proteins could differ biologically. Previously, it has been noted that FGF-1 and FGF-2 can oligomerize in the presence of HLGAGs. Therefore, we investigated whether FGF-1 and FGF-2 oligomerize by the same mechanism or by a different one. Through a combination of matrix-assisted laser desorption ionization mass spectrometry and chemical crosslinking, we show here that, under identical conditions, FGF-1 and FGF-2 differ in the degree and kind of oligomerization. Furthermore, an extensive analysis of FGF-1 and FGF-2 uncomplexed and HLGAG complexed crystal structures enables us to readily explain why FGF-2 forms sequential oligomers whereas FGF-1 forms only dimers. FGF-2, which possesses an interface capable of protein association, forms a translationally related oligomer, whereas FGF-1, which does not have this interface, forms only a symmetrically related dimer. Taken together, these data show that FGF-1 and FGF-2, despite their sequence homology, differ in their mechanism of oligomerization. (+info)
Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold.
(34/24796)
We demonstrate that the ligand pocket of a lipocalin from Pieris brassicae, the bilin-binding protein (BBP), can be reshaped by combinatorial protein design such that it recognizes fluorescein, an established immunological hapten. For this purpose 16 residues at the center of the binding site, which is formed by four loops on top of an eight-stranded beta-barrel, were subjected to random mutagenesis. Fluorescein-binding BBP variants were then selected from the mutant library by bacterial phage display. Three variants were identified that complex fluorescein with high affinity, exhibiting dissociation constants as low as 35.2 nM. Notably, one of these variants effects almost complete quenching of the ligand fluorescence, similarly as an anti-fluorescein antibody. Detailed ligand-binding studies and site-directed mutagenesis experiments indicated (i) that the molecular recognition of fluorescein is specific and (ii) that charged residues at the center of the pocket are responsible for tight complex formation. Sequence comparison of the BBP variants directed against fluorescein with the wild-type protein and with further variants that were selected against several other ligands revealed that all of the randomized amino acid positions are variable. Hence, a lipocalin can be used for generating molecular pockets with a diversity of shapes. We term this class of engineered proteins "anticalins." Their one-domain scaffold makes them a promising alternative to antibodies to create a stable receptor protein for a ligand of choice. (+info)
Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces.
(35/24796)
A general strategy is described for improving the binding properties of small-molecule ligands to protein targets. A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein. When the ligand of interest is presented to the target protein by the second protein, additional protein-protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event. We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide. This approach provides a way to modulate the potency and specificity of biologically active compounds. (+info)
Conformational changes in tertiary structure near the ligand binding site of an integrin I domain.
(36/24796)
For efficient ligand binding, integrins must be activated. Specifically, a conformational change has been proposed in a ligand binding domain present within some integrins, the inserted (I) domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333-1340]. This proposal remains controversial, however, despite extensive crystal structure studies on the I domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333-1340; Liddington, R. & Bankston, L. (1998) Structure (London) 6, 937-938; Qu, A. & Leahy, D. J. (1996) Structure (London) 4, 931-942; and Baldwin, E. T., Sarver, R. W., Bryant, G. L., Jr., Curry, K. A., Fairbanks, M. B., Finzel, B. C. , Garlick, R. L., Heinrikson, R. L., Horton, N. C. & Kelly, L. L. (1998) Structure (London) 6, 923-935]. By defining the residues present in the epitope of a mAb against the human Mac-1 integrin (alphaMbeta2, CD11b/CD18) that binds only the active receptor, we provide biochemical evidence that the I domain itself undergoes a conformational change with activation. This mAb, CBRM1/5, binds the I domain very close to the ligand binding site in a region that is widely exposed regardless of activation as judged by reactivity with other antibodies. The conformation of the epitope differs in two crystal forms of the I domain, previously suggested to represent active and inactive receptor. Our data suggests that conformational differences in the I domain are physiologically relevant and not merely a consequence of different crystal lattice interactions. We also demonstrate that the transition between the two conformational states depends on species-specific residues at the bottom of the I domain, which are proposed to be in an interface with another integrin domain, and that this transition correlates with functional activity. (+info)
Regional differences in the inhibition of mouse in vivo [3H]Ro 15-1788 binding reflect selectivity for alpha 1 versus alpha 2 and alpha 3 subunit-containing GABAA receptors.
(37/24796)
The benzodiazepines flunitrazepam, diazepam, and Ro 15-1788 and the beta-carboline DMCM bind with equivalent affinity to the benzodiazepine binding site of GABAA receptors containing different alpha subunits (i.e., alpha 1, alpha 2, alpha 3, or alpha 5); whereas, the triazolopyridazine CL 218,872 and imidazopyridine zolpidem have higher affinity for alpha 1 subunit-containing GABAA receptors. In the present study, the in vivo binding of [3H]Ro 15-1788 in mouse cerebellum and spinal cord was used to establish the occupancy of the benzodiazepine binding site of GABAA receptors containing primarily alpha 1 and alpha 2/alpha 3 subunits, respectively. Thus, the nonselective compounds flunitrazepam, diazepam, and DMCM all produced a similar inhibition of binding in cerebellum and spinal cord (respective ID50 values of 0.2 to 0.3 mg/kg, 2 mg/kg, and 10 mg/kg i.p.); whereas, the alpha 1 selective compounds CL 218,872 and zolpidem were more potent at inhibiting [3H]Ro 15-1788 binding in the cerebellum (ID50 values 4.5 mg/kg and 10 mg/kg i.p.) compared to the spinal cord (ID50 values 12 mg/kg and > 30 mg/kg i.p.). Thus, the reduction of in vivo f[3H]Ro 15-1788 binding in tissues containing alpha 1 and alpha 2/alpha 3 receptor populations reflects the in vitro affinities of subtype selective compounds and should help to interpret the behavioral profile of such compounds. (+info)
A functional model for O-O bond formation by the O2-evolving complex in photosystem II.
(38/24796)
The formation of molecular oxygen from water in photosynthesis is catalyzed by photosystem II at an active site containing four manganese ions that are arranged in di-mu-oxo dimanganese units (where mu is a bridging mode). The complex [H2O(terpy)Mn(O)2Mn(terpy)OH2](NO3)3 (terpy is 2,2':6', 2"-terpyridine), which was synthesized and structurally characterized, contains a di-mu-oxo manganese dimer and catalyzes the conversion of sodium hypochlorite to molecular oxygen. Oxygen-18 isotope labeling showed that water is the source of the oxygen atoms in the molecular oxygen evolved, and so this system is a functional model for photosynthetic water oxidation. (+info)
Binding of Cob(II)alamin to the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii. Identification of dimethylbenzimidazole as the axial ligand.
(39/24796)
The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5'-triphosphates to 2'-deoxynucleoside 5'-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2'-deoxy-2'-methylenecytidine 5'-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR. (+info)
Both group IB and group IIA secreted phospholipases A2 are natural ligands of the mouse 180-kDa M-type receptor.
(40/24796)
Snake venom and mammalian secreted phospholipases A2 (sPLA2s) have been associated with toxic (neurotoxicity, myotoxicity, etc.), pathological (inflammation, cancer, etc.), and physiological (proliferation, contraction, secretion, etc.) processes. Specific membrane receptors (M and N types) for sPLA2s have been initially identified with snake venom sPLA2s as ligands, and the M-type 180-kDa receptor was cloned from different animal species. This paper addresses the problem of the endogenous ligands of the M-type receptor. Recombinant group IB and group IIA sPLA2s from human and mouse species have been prepared and analyzed for their binding properties to M-type receptors from different animal species. Both mouse group IB and group IIA sPLA2s are high affinity ligands (in the 1-10 nM range) for the mouse M-type receptor. These two sPLA2s are expressed in the mouse tissues where the M-type receptor is also expressed, making it likely that both types of sPLA2s are physiological ligands of the mouse M-type receptor. This conclusion does not hold for human group IB and IIA sPLA2s and the cloned human M-type receptor. The two mouse sPLA2s have relatively high affinities for the mouse M-type receptor, but they can have much lower affinities for receptors from other animal species, indicating that species specificity exists for sPLA2 binding to M-type receptors. Caution should thus be exerted in avoiding mixing sPLA2s, cells, or tissues from different animal species in studies of the biological roles of mammalian sPLA2s associated with an action through their membrane receptors. (+info)