Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases

Sequence analysis of chloroplast and mitochondrial large subunit rRNA genes from over 75 green algae disclosed 28 new group I intron-encoded proteins carrying a single LAGLIDADG motif. These putative homing endonucleases form four subfamilies of homologous enzymes, with the members of each subfamily being encoded by introns sharing the same insertion site. We showed that four divergent endonucleases from the I-CreI subfamily cleave the same DNA substrates. Mapping of the 66 amino acids that are conserved among the members of this subfamily on the 3-dimensional structure of I-CreI bound to its recognition sequence revealed that these residues participate in protein folding, homodimerization, DNA recognition and catalysis. Surprisingly, only seven of the 21 I-CreI amino acids interacting with DNA are conserved, suggesting that I-CreI and its homologs use different subsets of residues to recognize the same DNA sequence. Our sequence comparison of all 45 single-LAGLIDADG proteins identified so far suggests that these proteins share related structures and that there is a weak pressure in each subfamily to maintain identical protein–DNA contacts. The high sequence variability we observed in the DNA-binding site of homologous LAGLIDADG endonucleases provides insight into how these proteins evolve new DNA specificity.

Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus

Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantities sufficient for structural characterization by cryoelectron microscopy and three-dimensional (3D) reconstruction. Two cDNAs were prepared and expressed in HEK293 cells, one encoding the wild-type RyR3 and the other encoding RyR3 containing glutathione S-transferase (GST) fused to its amino terminus (GST-RyR3). RyR3 was purified from detergent-solubilized transfected cells by affinity chromatography using 12.6-kDa FK506-binding protein in the form of a GST fusion as the affinity ligand. Purification of GST-RyR3 was achieved by affinity chromatography by using glutathione-Sepharose. Purified recombinant RyR3 and GST-RyR3 proteins exhibited high-affinity [3H]ryanodine binding that was sensitive to activation by Ca2+ and caffeine and to inhibition by Mg2+. 3D reconstructions of both recombinant RyR3 and GST-RyR3 appeared very similar to that of the native RyR3 purified from bovine diaphragm. Comparison of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the GST domains and, hence, the amino termini of the RyR3 subunits are located in the “clamp” structures that form the corners of the square-shaped cytoplasmic region of homotetrameric RyR3. This study describes the 3D reconstruction of a recombinant ryanodine receptor and it demonstrates the potential of this technology for characterizing functional and structural perturbations introduced by site-directed mutagenesis.

Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array

Atomic level structures have been determined for the soluble forms of several colicins and toxins, but the structural changes that occur after membrane binding have not been well characterized. Changes occurring in the transition from the soluble to membrane-bound state of the C-terminal 190-residue channel polypeptide of colicin E1 (P190) bound to anionic membranes are described. In the membrane-bound state, the α-helical content increases from 60–64% to 80–90%, with a concomitant increase in the average length of the helical segments from 12 to 16 or 17 residues, close to the length required to span the membrane bilayer in the open channel state. The average distance between helical segments is increased and interhelix interactions are weakened, as shown by a major loss of tertiary structure interactions, decreased efficiency of fluorescence resonance energy transfer from an energy donor on helix V of P190 to an acceptor on helix IX, and decreased resonance energy transfer at higher temperatures, not observed in soluble P190, implying freedom of motion of helical segments. Weaker interactions are also shown by a calorimetric thermal transition of low cooperativity, and the extended nature of the helical array is shown by a 3- to 4-fold increase in the average area subtended per molecule to 4,200 Å2 on the membrane surface. The latter, with analysis of the heat capacity changes, implies the absence of a developed hydrophobic core in the membrane-bound P190. The membrane interfacial layer thus serves to promote formation of a highly helical extended two-dimensional flexible net. The properties of the membrane-bound state of the colicin channel domain (i.e., hydrophobic anchor, lengthened and loosely coupled α-helices, and close association with the membrane interfacial layer) are plausible structural features for the state that is a prerequisite for voltage gating, formation of transmembrane helices, and channel opening.

Functional Hierarchy of Simultaneously Expressed Adhesion Receptors: Integrin α2β1 but Not CD44 Mediates MV3 Melanoma Cell Migration and Matrix Reorganization within Three-dimensional Hyaluronan-containing Collagen MatricesV⃞

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including β1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both β1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only β1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-β1 and anti-α2 integrin mAbs, whereas mAbs blocking CD44, α3, α5, α6, or αv integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of β1 integrins was not restored via CD44. Because α2β1-mediated migration was neither synergized nor replaced by CD44–HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.

Three-dimensional structure of poliovirus receptor bound to poliovirus

Poliovirus initiates infection by binding to its cellular receptor (Pvr). We have studied this interaction by using cryoelectron microscopy to determine the structure, at 21-Å resolution, of poliovirus complexed with a soluble form of its receptor (sPvr). This density map aided construction of a homology-based model of sPvr and, in conjunction with the known crystal structure of the virus, allowed delineation of the binding site. The virion does not change significantly in structure on binding sPvr in short incubations at 4°C. We infer that the binding configuration visualized represents the initial interaction that is followed by structural changes in the virion as infection proceeds. sPvr is segmented into three well-defined Ig-like domains. The two domains closest to the virion (domains 1 and 2) are aligned and rigidly connected, whereas domain 3 diverges at an angle of ≈60°. Two nodules of density on domain 2 are identified as glycosylation sites. Domain 1 penetrates the “canyon” that surrounds the 5-fold protrusion on the capsid surface, and its binding site involves all three major capsid proteins. The inferred pattern of virus–sPvr interactions accounts for most mutations that affect the binding of Pvr to poliovirus.

Sequencing of 3-O sulfate containing heparin decasaccharides with a partial antithrombin III binding site

Heparin- and heparan sulfate-like glycosaminoglycans (HLGAGs) represent an important class of molecules that interact with and modulate the activity of growth factors, enzymes, and morphogens. Of the many biological functions for this class of molecules, one of its most important functions is its interaction with antithrombin III (AT-III). AT-III binding to a specific heparin pentasaccharide sequence, containing an unusual 3-O sulfate on a N-sulfated, 6-O sulfated glucosamine, increases 1,000-fold AT-III's ability to inhibit specific proteases in the coagulation cascade. In this manner, HLGAGs play an important biological and pharmacological role in the modulation of blood clotting. Recently, a sequencing methodology was developed to further structure-function relationships of this important class of molecules. This methodology combines a property-encoded nomenclature scheme to handle the large information content (properties) of HLGAGs, with matrix-assisted laser desorption ionization MS and enzymatic and chemical degradation as experimental constraints to rapidly sequence picomole quantities of HLGAG oligosaccharides. Using the above property-encoded nomenclature-matrix-assisted laser desorption ionization approach, we found that the sequence of the decasaccharide used in this study is ΔU2SHNS,6SI2SHNS,6SI2SHNS,6SIHNAc,6SGHNS,3S,6S (±DDD4–7). We confirmed our results by using integral glycan sequencing and one-dimensional proton NMR. Furthermore, we show that this approach is flexible and is able to derive sequence information on an oligosaccharide mixture. Thus, this methodology will make possible both the analysis of other unusual sequences in HLGAGs with important biological activity as well as provide the basis for the structural analysis of these pharamacologically important group of heparin/heparan sulfates.

Phosphatidylinositol 3-kinase-γ activates Bruton’s tyrosine kinase in concert with Src family kinases

Bruton’s tyrosine kinase (Btk) is essential for normal B lymphocyte development and function. The activity of Btk is partially regulated by transphosphorylation within its kinase domain by Src family kinases at residue Tyr-551 and subsequent autophosphorylation at Tyr-223. Activation correlates with Btk association with cellular membranes. Based on specific loss of function mutations, the Btk pleckstrin homology (PH) domain plays an essential role in this activation process. The Btk PH domain can bind in vitro to several lipid end products of the phosphatidylinositol 3-kinase (PI 3-kinase) family including phosphatidylinositol 3,4,5-trisphosphate. Activation of Btk as monitored by elevation of phosphotyrosine content and a cellular transformation response was dramatically enhanced by coexpressing a weakly activated allele of Src (E378G) and the two subunits of PI 3-kinase-γ. This activation correlates with new sites of phosphorylation on Btk identified by two-dimensional phosphopeptide mapping. Activation of Btk was dependent on the catalytic activity of all three enzymes and an intact Btk PH domain and Src transphosphorylation site. These combined data define Btk as a downstream target of PI 3-kinase-γ and Src family kinases.

Clusters of charged residues in protein three-dimensional structures.

Statistically significant charge clusters (basic, acidic, or of mixed charge) in tertiary protein structures are identified by new methods from a large representative collection of protein structures. About 10% of protein structures show at least one charge cluster, mostly of mixed type involving about equally anionic and cationic residues. Positive charge clusters are very rare. Negative (or histidine-acidic) charge clusters often coordinate calcium, or magnesium or zinc ions [e.g., thermolysin (PDB code: 3tln), mannose-binding protein (2msb), aminopeptidase (1amp)]. Mixed-charge clusters are prominent at interchain contacts where they stabilize quaternary protein formation [e.g., glutathione S-transferase (2gst), catalase (8act), and fructose-1,6-bisphosphate aldolase (1fba)]. They are also involved in protein-protein interaction and in substrate binding. For example, the mixed-charge cluster of aspartate carbamoyl-transferase (8atc) envelops the aspartate carbonyl substrate in a flexible manner (alternating tense and relaxed states) where charge associations can vary from weak to strong. Other proteins with charge clusters include the P450 cytochrome family (BM-3, Terp, Cam), several flavocytochromes, neuraminidase, hemagglutinin, the photosynthetic reaction center, and annexin. In each case in Table 2 we discuss the possible role of the charge clusters with respect to protein structure and function.