Critical structure-function determinants within the N-terminal region of pulmonary surfactant protein SP-B

Surfactant protein SP-B is absolutely required for the surface activity of pulmonary surfactant and postnatal lung function. The results of a previous study indicated that the N-terminal segment of SP-B, comprising residues 1–9, is specifically required for surface activity, and suggested that prolines 2, 4, and 6 as well as tryptophan 9, may constitute essential structural motifs for protein function. In this work, we assessed the role of these two motifs in promoting the formation and maintenance of surface-active films. Three synthetic peptides were synthesized including a peptide corresponding to the N-terminal 37 amino acids of native SP-B and two variants in which prolines 2, 4, 6, or tryptophan 9 were substituted by alanines. All three synthetic peptides were surface-active, as expected from their amphipathic structure. The peptides were also able to insert into dipalmitoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol (7:3 w/w ratio) monolayers preformed at pressures >30 mN/m, indicating that they perturb and insert into membranes. Substitution of alanine for tryptophan at position 9 significantly decreased both the rate of adsorption/insertion of the peptide into the interface and reinsertion of surface-active material excluded from the film during successive compression-expansion cycles. Substitution of alanines for prolines at positions 2, 4, and 6 did not produce substantial changes in the rate of adsorption/insertion; however, reinsertion of surface-active material into the expanding interface film was not as effective as in the presence of the nativelike peptide. These results suggest that W9 is critical for optimal interface affinity, whereas prolines may promote a conformation that facilitates rapid insertion of the peptide into phospholipid monolayers compressed to the highest pressures during compression-expansion cycling.     

T cell recognition of nonpolymorphic determinants on H-2 class I molecules

Recognition of polymorphic determinants on class I or class II MHC Ag is required for T lymphocyte responses. Using cell-size artificial membranes (pseudocytes) bearing H-2 class I Ag it is demonstrated that T cells can, in addition, recognize nonpolymorphic determinants on class I proteins. Pseudocytes bearing class I alloantigen stimulate in vitro generation of secondary allogeneic CTL responses. At a suboptimal alloantigen surface density, incorporation of class I molecules identical to those of the responder cells (self-H-2) or from third-party cells resulted in dramatically enhanced responses, whereas incorporation of class II proteins had no effect. The receptor that mediates recognition of conserved class I determinants has not been identified, but results of antibody blocking studies are consistent with the Lyt-2/3 complex of CTL having this role. Thus, class I proteins on Ag-bearing cells can have two distinct roles in T cell activation, one involving recognition of polymorphic determinants by the Ag-specific receptor and the other involving recognition of conserved determinants.     

Structural determinants of SecB recognition by SecA in bacterial protein translocation

SecB is a bacterial chaperone involved in directing pre-protein to the translocation pathway by its specific interaction with the peripheral membrane ATPase SecA. The SecB-binding site on SecA is located at its C terminus and consists of a stretch of highly conserved residues. The crystal structure of SecB in complex with the C-terminal 27 amino acids of SecA from Haemophilus influenzae shows that the SecA peptide is structured as a CCCH zinc-binding motif. One SecB tetramer is bound by two SecA peptides, and the interface involves primarily salt bridges and hydrogen bonding interactions. The structure explains the importance of the zinc-binding motif and conserved residues at the C terminus of SecA in its high-affinity binding with SecB. It also suggests a model of SecB-SecA interaction and its implication for the mechanism of pre-protein transfer in bacterial protein translocation.     

Specificity determinants of substrate recognition by the protein kinase DYRK1A

DYRK1A is a dual-specificity protein kinase that is thought to be involved in brain development. We identified a single phosphorylated amino acid residue in the DYRK substrate histone H3 (threonine 45) by mass spectrometry, phosphoamino acid analysis, and protein sequencing. Exchange of threonine 45 for alanine abolished phosphorylation of histone H3 by DYRK1A and by the related kinases DYRK1B, DYRK2, and DYRK3 but not by CLK3. In order to define the consensus sequence for the substrate specificity of DYRK1A, a library of 300 peptides was designed in variation of the H3 phosphorylation site. Evaluation of the phosphate incorporation into these peptides identified DYRK1A as a proline-directed kinase with a phosphorylation consensus sequence (RPX(S/T)P) similar to that of ERK2 (PX(S/T)P). A peptide designed after the optimal substrate sequence (DYRKtide) was efficiently phosphorylated by DYRK1A (K(m) = 35 microM) but not by ERK2. Both ERK2 and DYRK1A phosphorylated myelin basic protein, whereas only ERK2, but not DYRK1A, phosphorylated the mitogen-activated protein kinase substrate ELK-1. This marked difference in substrate specificity between DYRK1A and ERK2 can be explained by the requirement for an arginine at the P -3 site of DYRK substrates and its presumed interaction with aspartate 247 conserved in all DYRKs.     

A new method for protein domain recognition

A fuzzy cluster method is presented to recognize protein domains. This algorithm can identify domains globally. A protein structure set was used to test the algorithm. Among 219 proteins, 66.7% yielded results that agreed with the reference definitions, 30.6% showed minor differences, and only 2.7% (six proteins) showed major differences with the reference. The new method is more than 20 times fast than previous algorithms. Received: 9 November 1998 / Revised version: 20 December 1999 / Accepted: 20 December 1999     

A structure-function study of ligand recognition by CD22beta

B-cell-specific CD22 is a member of a group of cell adhesion molecules within the immunoglobulin superfamily that display binding to glycans with terminal sialic acid residues. Binding of endogenous ligands to CD22 triggers B-cell activation and proliferation. It is therefore conceivable that high affinity ligands for CD22 may be of value as inhibitors of B-cell activation in allergy and chronic inflammation. In this study, we aimed to delineate the structural requirements for ligand binding to CD22. A library of 20 mono-, di-, and trisaccharide analogs of the basic binding motif Neu5Ac(alpha2,6)Lac was synthesized and screened for affinity for CD22beta. In general, CD22 ligand recognition appeared to be rather tolerant with respect to structural modifications of the anomeric sugar on a mono-, di-, and trisaccharide level, although affinity was increased by the presence of a nitro aromatic group at C-2. The most potent monovalent ligand, Neu5Ac-4-nitrobenzoyl-Glc, was selected to generate multivalent ligands based on either a glutamate or Tris cluster core. All multivalent ligands displayed at least a 10-fold increased affinity for CD22 compared with the corresponding monovalent glycoside. Interestingly, a maximal gain in affinity was already obtained for bivalent ligands, regardless of the terminal glycoside. A trivalent Tris-based cluster of Neu5Ac-4-nitrobenzoyl-Glc displayed a 300-fold higher affinity compared with the basic binding motif, which makes it, to our knowledge, the most potent antagonist for CD22 yet synthesized. As our in vitro fluorescence-activated cell sorting studies demonstrated efficient cellular uptake of a CD22 substrate, the most potent ligand in this study may hold promise as a homing device for immunomodulatory compounds and cytostatics.     

Mathematical modelling of charge properties of protein molecules]

A procedure of theoretical determination of the dependence of protein molecule charge on the medium pH has been developed. The suggested procedure allows calculating the protein pI value, the molecule charge at the definite pH value, as well as the corresponding values for the protein molecule. Calculations for insulin, apo A-I and apo A-II molecules have been carried out. Calculated pI values are equal to 5.25, 5.64 and 4.86, respectively. A comparison of the theoretical curves and experimental data allows obtaining information of the molecule structure. Carboxyl groups with abnormally high pK values are discovered, that, probably, indicates to the direct interaction of two COOH-groups. A supposition is made on the most probable arrangement of the functional fragments in apo A-I and apo A-II molecules.     

Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases

Two epidermal growth factor-stimulated protein kinases that correspond to ERK1 and ERK2 have been purified from human epidermoid carcinoma cells (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). A consensus primary sequence for substrates of ERK1 has been identified as -Pro-Leu-Ser/Thr-Pro- (Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991) J. Biol. Chem. 266, 15277-15285). However, the structural determinants for substrate recognition are not understood. We performed a systematic analysis of the effect of point mutations in the primary sequence of peptide substrates on the rate of phosphorylation by ERK1 and ERK2. The results of this investigation demonstrate that the substrate specificities of the ERK1 and ERK2 protein kinases are very similar. We propose that the primary sequence of substrates for ERK1 and ERK2 protein kinases can be generalized as -Pro-Xaan-Ser/Thr-Pro- (where Xaa is a neutral or basic amino acid and n = 1 or 2).     

Cellular phospholipases A2, structure-function relationship]

Two PLA2 are involved in cell signaling and in phospholipid homeostasis in mammalian cells. The first one is a 14 kDa protein whose cDNA was cloned in 1989. This enzyme exhibits a strong homology with pancreatic PLA2 but is more related to type II PLA2. This PLA2 is secreted by different tissues in response to inflammatory processes. Their main function seems to be the hydrolysis of membranes of altered cells or of bacteria and the stimulation of lipid mediator synthesis. The first cDNA of an another important PLA2 group was cloned in 1991. The protein deduced is a 88 kDa cytoplasmic protein. It is involved in cell signaling by stimulating the production of free fatty acids and of their oxygenated products. These products might in turn either activate transducing proteins or stimulate membrane receptors.     

Computational method for prediction of protein functional sites using specificity determinants

The current available data on protein sequences largely exceeds the experimental capabilities to annotate their function. So annotation in silico, i.e. using computational methods becomes increasingly important. This annotation is inevitably a prediction, but it can be an important starting point for further experimental studies. Here we present a method for prediction of protein functional sites, SDPsite, based on the identification of protein specificity determinants. Taking as an input a protein sequence alignment and a phylogenetic tree, the algorithm predicts conserved positions and specificity determinants, maps them onto the protein's 3D structure, and searches for clusters of the predicted positions. Comparison of the obtained predictions with experimental data and data on performance of several other methods for prediction of functional sites reveals that SDPsite agrees well with the experiment and outperforms most of the previously available methods. SDPsite is publicly available under http://bioinf.fbb.msu.ru/SDPsite.     

DNA recognition by quinoline antibiotics: use of base-modified DNA molecules to investigate determinants of sequence-specific binding of luzopeptin

The luzopeptin antibiotics contain a cyclic decadepsipeptide to which are attached two quinoline chromophores that bisintercalate into DNA. Although they bind DNA less tightly than the structurally related quinoxaline antibiotics echinomycin and triostin A, the molecular basis of their interaction remains unclear. We have used the PCR in conjunction with novel nucleotides to create specifically modified DNA for footprinting experiments. In order to study the influence that removal, addition or relocation of the guanine 2-amino group, which normally identifies G.C base pairs from the minor groove, has on the interaction of luzopeptin antibiotics with DNA. The presence of a purine 2-amino group is not strictly required for binding of luzopeptin to DNA, but the exact location of this group can alter the position of preferred drug binding sites. It is, however, not the sole determinant of nucleotide sequence recognition in luzopeptin-DNA interaction. Nor can the selectivity of luzopeptin be attributed to the quinoline chromophores, suggesting that an analogue mode of DNA recognition may be operative. This is in contrast to the digital readout that seems to predominate with the quinoxaline antibiotics.     

Structural and dynamic determinants of protein-peptide recognition

Protein-peptide interactions play important roles in many cellular processes, including signal transduction, trafficking, and immune recognition. Protein conformational changes upon binding, an ill-defined peptide binding surface, and the large number of peptide degrees of freedom make the prediction of protein-peptide interactions particularly challenging. To address these challenges, we perform rapid molecular dynamics simulations in order to examine the energetic and dynamic aspects of protein-peptide binding. We find that, in most cases, we recapitulate the native binding sites and native-like poses of protein-peptide complexes. Inclusion of electrostatic interactions in simulations significantly improves the prediction accuracy. Our results also highlight the importance of protein conformational flexibility, especially side-chain movement, which allows the peptide to optimize its conformation. Our findings not only demonstrate the importance of sufficient sampling of the protein and peptide conformations, but also reveal the possible effects of electrostatics and conformational flexibility on peptide recognition.     

Sequence and structural determinants of mannose recognition

Mannose, an abundant cell surface monosaccharide binds to a diverse set of receptors, which are involved in a variety of important cellular processes. Structural analysis has been carried out on all the proteins containing non-covalently bound mannose as a monosaccharide in the Protein Data Bank, to identify common recognition principles. Proteins, highly specific to mannose, belonging to the super family of bulb lectins, are found to contain a consensus sequence motif QXDXNXVXY, which has been identified to be essential for mannose binding. Analysis of this motif in the crystal structures of bulb lectins has led to the understanding of the contribution of individual residues in mannose recognition. Comparison with other mannose binding proteins, reveals common hydrogen bonding patterns in all of them, despite differences in sequence, overall fold and the substructures at the binding sites of individual proteins. A database analysis also suggests that although the topology of the backbone, as at the binding site in bulb lectins, can generate mannose binding capability in a few other proteins, sequence and disposition of not only the residues in the motif, but also the residues in the neighborhood play a crucial role in allowing that property to be retained.     

Structural determinants of integrin recognition by talin

The binding of cytoplasmic proteins, such as talin, to the cytoplasmic domains of integrin adhesion receptors mediates bidirectional signal transduction. Here we report the crystal structure of the principal integrin binding and activating fragment of talin, alone and in complex with fragments of the beta 3 integrin tail. The FERM (four point one, ezrin, radixin, and moesin) domain of talin engages integrins via a novel variant of the canonical phosphotyrosine binding (PTB) domain-NPxY ligand interaction that may be a prototype for FERM domain recognition of transmembrane receptors. In combination with NMR and mutational analysis, our studies reveal the critical interacting elements of both talin and the integrin beta 3 tail, providing structural paradigms for integrin linkage to the cell interior.