Essential role of B-helix calcium binding sites in annexin V-membrane binding

Crystal structures of annexin V have shown up to 10 bound calcium ions in three different types of binding sites, but previous work concluded that only one of these sites accounted for nearly all of the membrane binding affinity of the molecule. In this study we mutated residues contributing to potential calcium binding sites in the AB and B helices in each of the four domains (eight sites in total) and in DE helices in the first, second, and third domains (three sites in total). We measured the affinity of each protein for phospholipid vesicles and cell membranes by quantitative calcium titration under low occupancy conditions (< 1% saturation of available membrane binding sites). Affinity was calculated from the midpoint and slope of the calcium titration curve and the concentration of membrane binding sites. The results showed that all four AB sites were essential for high affinity binding, as were three of the four B sites (in domains 1, 2, and 3); the DE site in the first domain made a slight contribution to affinity. Multisite mutants showed that each domain contributed additively and independently to binding affinity; in contrast, AB and B sites within the same domain were interdependent. The number of functionally important sites identified was consistent with the Hill coefficient observed in calcium titrations. This study shows an essential and previously unappreciated role for B-helix calcium binding sites in the membrane binding of annexins and indicates that all four domains of the molecule are required for maximum membrane binding affinity.     

Allosteric Communication Occurs via Networks of Tertiary and Quaternary Motions in Proteins

Allosteric proteins bind an effector molecule at one site resulting in a functional change at a second site. We hypothesize that allosteric communication in proteins relies upon networks of quaternary (collective, rigid-body) and tertiary (residue–residue contact) motions. We argue that cyclic topology of these networks is necessary for allosteric communication. An automated algorithm identifies rigid bodies from the displacement between the inactive and the active structures and constructs “quaternary networks” from these rigid bodies and the substrate and effector ligands. We then integrate quaternary networks with a coarse-grained representation of contact rearrangements to form “global communication networks” (GCNs). The GCN reveals allosteric communication among all substrate and effector sites in 15 of 18 multidomain and multimeric proteins, while tertiary and quaternary networks exhibit such communication in only 4 and 3 of these proteins, respectively. Furthermore, in 7 of the 15 proteins connected by the GCN, 50% or more of the substrate-effector paths via the GCN are “interdependent” paths that do not exist via either the tertiary or the quaternary network. Substrate-effector “pathways” typically are not linear but rather consist of polycyclic networks of rigid bodies and clusters of rearranging residue contacts. These results argue for broad applicability of allosteric communication based on structural changes and demonstrate the utility of the GCN. Global communication networks may inform a variety of experiments on allosteric proteins as well as the design of allostery into non-allosteric proteins.     

Sequences adjacent to the 5' splice site control U1A binding upstream of the IgM heavy chain secretory poly(A) site

We have recently shown that the stability of the alternatively expressed immunoglobulin M heavy chain secretory mRNA is developmentally regulated by U1A. U1A binds novel non-consensus sites upstream of the secretory poly(A) site and inhibits poly(A) tail addition in undifferentiated cells. U1A's dependence for binding and function upon a stem-loop structure has been extensively characterized for the consensus sites. We therefore probed the structure surrounding the novel U1A binding sites. We show that two of the three novel binding sites represent the major single-stranded regions upstream of the secretory poly(A) site, consistent with a major role at this site. The strength of binding and ability of U1A to inhibit poly(A) polymerase correlate with the accessibility of the novel sites. However, long range interactions are responsible for maintaining them in an open configuration. Mutation of an RNase V1-sensitive site 102 nucleotides upstream, directly adjacent to the competing 5' splice site, changes the structure of one the U1A binding sites and thus abolishes the binding of the second U1A molecule and the ability of U1A ability to inhibit poly(A) polymerase activity at this site. These sites bind U1A via its N-terminal domain but with a 10-fold lower affinity than U1 small nuclear RNA. This lower binding affinity is more conducive to U1A's regulation of poly(A) tail addition to heterologous mRNA.     

Neutralization epitopes of the F glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function.

Eighteen neutralizing monoclonal antibodies (MAbs) specific for the fusion glycoprotein of the A2 strain of respiratory syncytial virus (RSV) were used to construct a detailed topological and operational map of epitopes involved in neutralization and fusion. Competitive binding assays identified three nonoverlapping antigenic sites (A, B, and C) and one bridge site (AB). Thirteen MAb-resistant mutants (MARMs) were selected, and the neutralization patterns of the MAbs with either MARMs or RSV clinical strains identified a minimum of 16 epitopes. MARMs selected with antibodies to six of the site A and AB epitopes displayed a small-plaque phenotype, which is consistent with an alteration in a biologically active region of the F molecule. Analysis of MARMs also indicated that these neutralization epitopes occupy topographically distinct but conformationally interdependent regions with unique biological and immunological properties. Antigenic variation in F epitopes was examined by using 23 clinical isolates (18 subgroup A and 5 subgroup B) in cross-neutralization assays with the 18 anti-F MAbs. This analysis identified constant, variable, and hypervariable regions on the molecule and indicated that antigenic variation in the neutralization epitopes of the RSV F glycoprotein is the result of a noncumulative genetic heterogeneity. Of the 16 eptiopes, 8 were conserved on all or all but 1 of 23 subgroup A or subgroup B clinical isolates.     

Allosteric control of three B12-dependent (class II) ribonucleotide reductases. Implications for the evolution of ribonucleotide reduction

Three separate classes of ribonucleotide reductases are known, each with a distinct protein structure. One common feature of all enzymes is that a single protein generates each of the four deoxyribonucleotides. Class I and III enzymes contain an allosteric substrate specificity site capable of binding effectors (ATP or various deoxyribonucleoside triphosphates) that direct enzyme specificity. Some (but not all) enzymes contain a second allosteric site that binds only ATP or dATP. Binding of dATP to this site inhibits the activity of these enzymes. X-ray crystallography has localized the two sites within the structure of the Escherichia coli class I enzyme and identified effector-binding amino acids. Here, we have studied the regulation of three class II enzymes, one from the archaebacterium Thermoplasma acidophilum and two from eubacteria (Lactobacillus leichmannii and Thermotoga maritima). Each enzyme has an allosteric site that binds ATP or various deoxyribonucleoside triphosphates and that regulates its substrate specificity according to the same rules as for class I and III enzymes. dATP does not inhibit enzyme activity, suggesting the absence of a second active allosteric site. For the L. leichmannii and T. maritima enzymes, binding experiments also indicate the presence of only one allosteric site. Their primary sequences suggest that these enzymes lack the structural requirements for a second site. In contrast, the T. acidophilum enzyme binds dATP at two separate sites, and its sequence contains putative effector-binding amino acids for a second site. The presence of a second site without apparent physiological function leads to the hypothesis that a functional site was present early during the evolution of ribonucleotide reductases, but that its function was lost from the T. acidophilum enzyme. The other two B12 enzymes lost not only the function, but also the structural basis for the site. Also a large subgroup (Ib) of class I enzymes, but none of the investigated class III enzymes, has lost this site. This is further indirect evidence that class II and I enzymes may have arisen by divergent evolution from class III enzymes.     

Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase

The mammalian cAMP-dependent protein kinases have regulatory (R) subunits that show substantial homology in amino acid sequence with the catabolite gene activator protein (CAP), a cAMP-dependent gene regulatory protein from Escherichia coli. Each R subunit has two in-tandem cAMP binding domains, and the structure of each of these domains has been modeled by analogy with the crystal structure of CAP. Both the type I and II regulatory subunits have been considered, so that four cAMP binding domains have been modeled. The binding of cAMP in general is analogous in all the structures and has been correlated with previous results based on photolabeling and binding of cAMP analogues. The model predicts that the first cAMP binding domain correlates with the previously defined fast dissociation site, which preferentially binds N6-substituted analogues of cAMP. The second domain corresponds to the slow dissociation site, which has a preference for C8-substituted analogues. The model also is consistent with cAMP binding in the syn conformation in both sites. Finally, this model has targeted specific regions that are likely to be involved in interdomain contacts. This includes contacts between the two cAMP binding domains as well as contacts with the amino-terminal region of the R subunit and with the catalytic subunit.     

Nonspecific base recognition mediated by water bridges and hydrophobic stacking in ribonuclease I from Escherichia coli

The crystal structure of Escherichia coli ribonuclease I (EcRNase I) reveals an RNase T2-type fold consisting of a conserved core of six beta-strands and three alpha-helices. The overall architecture of the catalytic residues is very similar to the plant and fungal RNase T2 family members, but the perimeter surrounding the active site is characterized by structural elements specific for E. coli. In the structure of EcRNase I in complex with a substrate-mimicking decadeoxynucleotide d(CGCGATCGCG), we observe a cytosine bound in the B2 base binding site and mixed binding of thymine and guanine in the B1 base binding site. The active site residues His55, His133, and Glu129 interact with the phosphodiester linkage only through a set of water molecules. Residues forming the B2 base recognition site are well conserved among bacterial homologs and may generate limited base specificity. On the other hand, the B1 binding cleft acquires true base aspecificity by combining hydrophobic van der Waals contacts at its sides with a water-mediated hydrogen-bonding network at the bottom. This B1 base recognition site is highly variable among bacterial sequences and the observed interactions are unique to EcRNaseI and a few close relatives.     

Allosteric changes in solvent accessibility observed in thrombin upon active site occupation

The solvent accessibility of thrombin in its substrate-free and substrate-bound forms has been compared by amide hydrogen/deuterium (H/(2)H) exchange. The optimized inhibitor peptide dPhe-Pro-Arg chloromethyl ketone (PPACK) was used to simulate the substrate-bound form of thrombin. These studies were motivated by the lack of observed changes in the active site of thrombin in the crystal structure of the thrombin-thrombomodulin complex. This result appeared to contradict amide exchange studies on the thrombin-thrombomodulin complex that suggested subtle changes occur in the active site loops upon thrombomodulin binding. Our results show that two active site loops, residues 214-222 and residues 126-132, undergo decreases in solvent accessibility due to steric contacts with PPACK substrate. However, we also observe two regions outside the active site undergoing solvent protection upon substrate binding. The first region corresponds to anion binding exosite 1, and the second is a beta-strand-containing loop which runs through the core of the molecule and contains Trp141 which makes critical contacts with anion binding exosite 1. These results indicate two pathways of allosteric change that connect the active site to the distal anion binding exosite 1.     

Interlobe communication in multiple calcium-binding site mutants of Drosophila calmodulin.

We have generated mutants of Drosophila calmodulin in which pairs of calcium-binding sites are mutated so as to prevent calcium binding. In all sites, the mutation involves replacement of the -Z position glutamate residue with glutamine. Mutants inactivated in both N-terminal sites (B12Q) or both C-terminal sites (B34Q), and two mutants with one N- and one C-terminal site inactivated (B13Q and B24Q) were generated. The quadruple mutant with all four sites mutated was also studied. UV-difference spectroscopy and near-UV CD were used to examine the influence of these mutations upon the single tyrosine (Tyr-138) of the protein. These studies uncovered four situations in which Tyr-138 in the C-terminal lobe responds to a change to the calcium-binding properties of the N-terminal lobe. Further, they suggest that N-terminal calcium-binding events contribute strongly to the aberrant behavior of Tyr-138 seen in mutants with a single functional C-terminal calcium-binding site. The data also indicate that loss of calcium binding at site 1 adjusts the aberrant conformation of Tyr-138 produced by mutation of site 3 toward the wild-type structure. However, activation studies for skeletal muscle myosin light chain kinase (SK-MLCK) established that all of the multiple binding site mutants are poor activators of SK-MLCK. Thus, globally, the calcium-induced conformation of B13Q is not closer to wild type than that of either the site 1 or the site 3 mutant. The positioning of Tyr-138 within the crystal structure of calmodulin suggests that effects of the N-terminal lobe on this residue may be mediated via changes to the central linker region of the protein.     

A second binding site revealed by C-terminal truncation of calpain small subunit, a penta-EF-hand protein

The subunits in calpain and in the related penta-EF-hand (PEF) proteins are bound through contacts between the unpaired EF-hand 5 from each subunit. To study subunit binding further, a tetra-EF-hand 18 kDa N- and C-terminally truncated form of the calpain small subunit was prepared (18k). This protein does not combine with the calpain large subunit to form active calpain, but forms homodimers in solution, as shown by ultracentrifugation. The X-ray structure of the 18k protein in the presence of cadmium was solved to a resolution of 2.0 A. The structure of the monomer is almost identical to the known structure of the calpain small subunit, but the 18k protein forms an oligomer in the crystal by the use of two binding sites. One of these sites is an artefact arising from the C-terminal truncation, but the other is a naturally occurring site that is fully exposed to water in intact purified calpain. The characteristics of this site suggest that it may be important in binding other protein modulators involved in the regulation of calpain and of PEF proteins.     

Mechanism of transducin activation of frog rod photoreceptor phosphodiesterase. Allosteric interactiona between the inhibitory gamma subunit and the noncatalytic cGMP-binding sites

The rod photoreceptor phosphodiesterase (PDE) is unique among all known vertebrate PDE families for several reasons. It is a catalytic heterodimer (alphabeta); it is directly activated by a G-protein, transducin; and its active sites are regulated by inhibitory gamma subunits. Rod PDE binds cGMP at two noncatalytic sites on the alphabeta dimer, but their function is unclear. We show that transducin activation of frog rod PDE introduces functional heterogeneity to both the noncatalytic and catalytic sites. Upon PDE activation, one noncatalytic site is converted from a high affinity to low affinity state, whereas the second binding site undergoes modest decreases in binding. Addition of gamma to transducin-activated PDE can restore high affinity binding as well as reducing cGMP exchange kinetics at both sites. A strong correlation exists between cGMP binding and gamma binding to activated PDE; dissociation of bound cGMP accompanies gamma dissociation from PDE, whereas addition of either cGMP or gamma to alphabeta dimers can restore high affinity binding of the other molecule. At the active site, transducin can activate PDE to about one-half the turnover number for catalytic alphabeta dimers completely lacking bound gamma subunit. These results suggest a mechanism in which transducin interacts primarily with one PDE catalytic subunit, releasing its full catalytic activity as well as inducing rapid cGMP dissociation from one noncatalytic site. The state of occupancy of the noncatalytic sites on PDE determines whether gamma remains bound to activated PDE or dissociates from the holoenzyme, and may be relevant to light adaptation in photoreceptor cells.     

Structures of in vitro evolved binding sites on neocarzinostatin scaffold reveal unanticipated evolutionary pathways

We have recently applied in vitro evolution methods to create in Neocarzinostatin a new binding site for a target molecule unrelated to its natural ligand. The main objective of this work was to solve the structure of some of the selected binders in complex with the target molecule: testosterone. Three proteins (1a.15, 3.24 and 4.1) were chosen as representative members of sequence families that came out of the selection process within different randomization schemes. In order to evaluate ligand-induced conformational adaptation, we also determined the structure of one of the proteins (3.24) in the free and complexed forms. Surprisingly, all these mutants bind not one but two molecules of testosterone in two very different ways. The 3.24 structure revealed that the protein spontaneously evolved in the system to bind two ligand molecules in one single binding crevice. These two binding sites are formed by substituted as well as by non-variable side-chains. The comparison with the free structure shows that only limited structural changes are observed upon ligand binding. The X-ray structures of the complex formed by 1a.15 and 4.1 Neocarzinostatin mutants revealed that the two variants form very similar dimers. These dimers were observed neither for the uncomplexed variants nor for wild-type Neocarzinostatin but were shown here to be induced by ligand binding. Comparison of the three complexed forms clearly suggests that these unanticipated structural responses resulted from the molecular arrangement used for the selection experiments.     

Different functional sites on rIFN-alpha 2 and their relation to the cellular receptor binding site

Functional domains on the recombinant interferon-alpha 2 (rIFN-alpha 2) molecule, which are involved in antiviral and NK enhancing activities, have been defined by immunochemical mapping with MAb, and their relationship with the IFN cellular receptor binding site has been studied. With 20 different anti-IFN-alpha 2 MAb selected by their binding to 125I-labeled IFN and by immunoprecipitation of the 20 Kd IFN molecule, we have defined three spatially separated epitopes (designated as sites A, B, and C) and two partially overlapping antigenic determinants on the IFN-alpha 2 molecule. Functional relation of IFN-alpha 2 A, B, and C epitopes have been determined by assaying the effect of various anti-IFN MAb on IFN-mediated biologic activities. MAb directed to sites A and B neutralized the antiviral activity of IFN. Furthermore, the MAb specific for site B displayed a neutralizing potency threefold higher than MAb directed to site A. Site B was also involved in the enhancing activity of IFN on NK-mediated cell cytotoxicity, whereas site A was not. MAb directed to site C partially affected the IFN-boosted NK activity but did not neutralize the IFN antiviral activity. Inhibition studies of 125I-IFN binding to human U-937 myelomonocytic cells by anti-IFN MAb demonstrated that MAb directed to site B blocked different IFN biologic functions by preventing its binding to the cellular receptor, whereas MAb directed to sites A and C caused no inhibition and partial inhibition of this binding, respectively.     

Mutational analysis of protein binding sites involved in formation of the bacteriophage lambda attL complex.

Bacteriophage lambda site-specific recombination requires the formation of higher-order protein-DNA complexes to accomplish synapsis of the partner attachment (att) sites as well as for the regulation of the integration and excision reactions. The att sites are composed of a core region, the actual site of strand exchange, and flanking arm regions. The attL site consists of two core sites (C and C'), an integration host factor (IHF) binding site (H'), and three contiguous Int binding arm sites (P'1, P'2, and P'3). In this study, we employed bacteriophage P22 challenge phages to determine which protein binding sites participate in attL complex formation in vivo. The C', H', and P'1 sites were critical, because mutations in these sites severely disrupted formation of the attL complex. Mutations in the C and P'2 sites were less severe, and alteration of the P'3 site had no effect on complex formation. These results support a model in which IHF, bound to the H' site, bends the attL DNA so that the Int molecule bound to P'1 also interacts with the C' core site. This bridged complex, along with a second Int molecule bound to P'2, helps to stabilize the interaction of a third Int with the C core site. The results also indicate that nonspecific DNA binding is a significant component of the Int-core interactions and that the cooperativity of Int binding can overcome the effects of mutations in the individual arm sites and core sites.     

Diversity of B2 bradykinin receptors with nanomolar affinity expressed in passaged IMR90 human lung fibroblasts.

IMR90 human fetal lung fibroblasts express bradykinin receptors activating the pathway for biosynthesis of PGE2. A receptor of the B2 subtype stimulates half-maximal PGE2 production at 4.8 nM bradykinin, and maximal output takes place at 25 nM bradykinin. Radioligand binding studies reveal a population of [3H]bradykinin binding sites whose affinity correlates with this B2 receptor's biologic activity, with a KD of 2.5 nM. As IMR90 cells reach 60% of their defined life span in culture, they spontaneously induce expression of a second site of lower affinity, with half-maximal binding of [3H]bradykinin at 44 nM. This second site displays a characteristic primary B2 receptor recognition profile, but differs from the 2.5 nM site on a secondary level in recognition among different B2 ligands. Bradykinin is the most potent ligand at both sites; they each preferentially recognize an N-terminal extended bradykinin peptide construct having selectivity for the rat myometrial B2 receptor, suggesting that both sites have structural features in common. However, they display diversity in their order of preference for Met-Lys-bradykinin versus Lys-Lys-bradykinin; at the 44 nM site this order is completely reversed from the order of potency exhibited at the 2.5 nM site. Expression of the second site changes the manner in which these fibroblasts control their PGE2 production; it affords a graded response of PGE2 production at bradykinin levels beyond those which would normally saturate the 2.5 nM site. The inducibility of the 44 nM site in cultured fibroblasts addresses in vivo conditions in an inflammatory environment where continuing generation of bradykinin-related peptides takes place and presents a possible mechanism for overriding constraints that would otherwise limit the progression of inflammation.     

Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication

Polyomavirus large T antigen binds to multiple 5′-G(A/G)GGC-3′ pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a “handover” mechanism mediated by these protein-protein contacts.     

Zinc coordination sphere in biochemical zinc sites

Zinc is known to be indispensable to growth and development and transmission of the genetic message. It does this through a remarkable mosaic of zinc binding motifs that orchestrate all aspects of metabolism. There are now nearly 200 three dimensional structures for zinc proteins, representing all six classes of enzymes and covering a wide range of phyla and species. These structures provide standards of reference for the identity and nature of zinc ligands in other proteins for which only the primary structure is known. Three primary types of zinc sites are apparent from examination of these structures: structural, catalytic and cocatalytic. The most common amino acids that supply ligands to these sites are His, Glu, Asp and Cys. In catalytic sites zinc generally forms complexes with water and any three nitrogen, oxygen and sulfur donors with His being the predominant amino acid chosen. Water is always a ligand to such sites. Structural zinc sites have four protein ligands and no bound water molecule. Cys is the preferred ligand in such sites. Cocatalytic sites contain two or three metals in close proximity with two of the metals bridged by a side chain moiety of a single amino acid residue, such as Asp, Glu or His and sometimes a water molecule. Asp and His are the preferred amino acids for these sites. No Cys ligands are found in such sites. The scaffolding of the zinc sites is also important to the function and reactivity of the bound metal. The influence of zinc on quaternary protein structure has led to the identification of a fourth type of zinc binding site, protein inteface. In this case zinc sites are formed from ligands supplied from amino acid residues residing in the binding surface of two proteins. The resulting zinc site usually has the coordination properties of a catalytic or structural zinc binding site.