Two of the five zinc fingers in the Zap1 transcription factor DNA binding domain dominate site-specific DNA binding.

The Zap1 transcriptional activator from Saccharomyces cerevisiae induces expression of a series of genes containing an 11 base pair conserved promoter element (ZRE) under conditions of zinc deficiency. This work shows that Zap1 uses four of its seven zinc finger domains to contact the ZRE and that two of these dominate the interaction by contacting the essential ACC-GGT ends. Two Zn finger domains (ZF1 and ZF2) do not contact DNA, and a third ZF3 may be more important for interfinger protein-protein interactions. Zn finger domains important for ZRE contact were identified from triple mutations in Zap1, changing three residues in the alpha helix in each finger known to be important for DNA contacts in Zn finger proteins. Replacement of -1, 3, and 6 helix residues in ZF4 and ZF7 reduced the affinity of Zap1 for the wild-type ZRE. In contrast, triple mutations within the intervening ZF5 and ZF6 domains had minimal effect. The data argue that fingers 4 and 7 contact the ACC-GGT ends while fingers 5 and 6 contact the 5 bp central ZRE sequence. This conclusion is corroborated by decreased Zap1 affinity for a ZRE DNA duplex containing mutations of the AC-GT ends of the ZRE, whereas transversion mutations within the central 5 bp of the ZRE had minimal effect on Zap1 binding affinity.

The ABRF-MIRG'02 study: assembly state, thermodynamic, and kinetic analysis of an enzyme/inhibitor interaction.

Fully characterizing the interactions involving biomolecules requires information on the assembly state, affinity, kinetics, and thermodynamics associated with complex formation. The analytical technologies often used to measure biomolecular interactions include analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). In order to evaluate the capabilities of core facilities to implement these technologies, the Association of Biomolecular Resource Facilities (ABRF) Molecular Interactions Research Group (MIRG) developed a standardized model system and distributed it to a panel of AUC, ITC, and SPR operators. The model system was composed of a well-characterized enzyme-inhibitor pair, namely bovine carbonic anhydrase II (CA II) and 4-carboxybenzenesulfonamide (CBS). Study participants were asked to measure one or more of the following: (1) the molecular mass, homogeneity, and assembly state of CA II by AUC; (2) the affinity and thermodynamics for complex formation by ITC; and (3) the affinity and kinetics of complex formation by SPR. The results from this study provide a benchmark for comparing the capabilities of individual laboratories and for defining the utility of the different instrumentation.

Current and emerging commercial optical biosensors.

The field of commercial optical biosensors is rapidly evolving, with new systems and detection methods being developed each year. This review outlines the currently available biosensor hardware and highlights unique features of each platform. Affinity-based biosensor technology, with its high sensitivity, wide versatility and high throughput, is playing a significant role in basic research, pharmaceutical development, and the food and environmental sciences. Likewise, the increasing popularity of biosensors is prompting manufacturers to develop new instrumentation for dedicated applications. We provide a preview of some of the emerging commercial systems that are dedicated to drug discovery, proteomics, clinical diagnostics and routine biomolecular interaction analysis.

Survey of the year 2000 commercial optical biosensor literature.

We have compiled a comprehensive list of the articles published in the year 2000 that describe work employing commercial optical biosensors. Selected reviews of interest for the general biosensor user are highlighted. Emerging applications in areas of drug discovery, clinical support, food and environment monitoring, and cell membrane biology are emphasized. In addition, the experimental design and data processing steps necessary to achieve high-quality biosensor data are described and examples of well-performed kinetic analysis are provided.

Biochemical analyses of the interactions between human immunodeficiency virus type 1 Vpr and p6(Gag).

The nonstructural human immunodeficiency virus type 1 Vpr protein is packaged into progeny virions at significant levels (approximately 200 copies/virion). Genetic analyses have demonstrated that efficient Vpr packaging is dependent upon a leucine-X-X-leucine-phenylalanine (LXXLF) motif located in the p6(Gag) domain of the structural Gag polyprotein. Recombinant proteins spanning full-length Vpr (Vpr(1-97)) or the amino-terminal 71 amino acids (Vpr(1-71)) formed specific complexes with recombinant p6 proteins in vitro. Complex formation required an intact LXXLF motif and exhibited an intrinsic dissociation constant of approximately 75 microM. Gel filtration and cross-linking analyses further revealed that Vpr(1-71) self-associated in solution. Our experiments demonstrate that Vpr can bind directly and specifically to p6 and suggest that oligomerization of both Vpr and Gag may serve to increase the avidity and longevity of Vpr-Gag complexes, thereby ensuring efficient Vpr packaging.

Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.

Like other enveloped viruses, HIV-1 uses cellular machinery to bud from infected cells. We now show that Tsg101 protein, which functions in vacuolar protein sorting (Vps), is required for HIV-1 budding. The UEV domain of Tsg101 binds to an essential tetrapeptide (PTAP) motif within the p6 domain of the structural Gag protein and also to ubiquitin. Depletion of cellular Tsg101 by small interfering RNA arrests HIV-1 budding at a late stage, and budding is rescued by reintroduction of Tsg101. Dominant negative mutant Vps4 proteins that inhibit vacuolar protein sorting also arrest HIV-1 and MLV budding. These observations suggest that retroviruses bud by appropriating cellular machinery normally used in the Vps pathway to form multivesicular bodies.

High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE.

Characterizing how chemical compounds bind to human serum albumin (HSA) is essential in evaluating drug candidates. Using warfarin as a test system, we validate the application of BIACORE SPR biosensors to reliably determine binding constants for drug/HSA interactions. The binding responses for warfarin over HSA surfaces were extremely reproducible even though warfarin is small compared to the size of the immobilized protein. At high concentrations, warfarin bound at more than one site on HSA, which is consistent with its known binding properties. The affinity we determined for the high-affinity site (K(25 degrees C)(d) = 3.7 +/- 1.2 microM), as well as the dissociation rate constant (k(25 degrees C)(d) = 1.2 s(-1)), are also consistent with binding constants determined previously. These results validate the biosensor technology and illustrate how BIACORE can be used to study drug/HSA interactions in a high-resolution mode. Using a set of 10 test compounds, we present a protocol for determining equilibrium dissociation constants for HSA in a high-throughput mode. Our method involves working at low compound concentrations and fitting the equilibrium data for all compounds simultaneously. We show that the % bound values determined by SPR correlate with the values determined by solution-based methods. The ability to examine directly the binding of small molecules (130-800 Da), coupled with minimal sample requirements and automated instrumentation, makes BIACORE technology applicable for evaluating drug/HSA interactions.

BIACORE J: a new platform for routine biomolecular interaction analysis.

SPR biosensor technology continues to evolve. The recently released platform from Biacore AB (Uppsala, Sweden), BIACORE J, is designed for the routine analysis of biomolecular interactions. Using an antibody-protein A and a ligand-receptor system, we demonstrate the utility of BIACORE J in determining active concentration and binding affinities. The results from these studies illustrate the high sensitivity of the instrument and its ability to generate reproducible binding responses. The BIACORE J is easy to operate and useful in diverse applications, making SPR technology widely accessible as a research tool.

G-protein signaling through tubby proteins.

Dysfunction of the tubby protein results in maturity-onset obesity in mice. Tubby has been implicated as a transcription regulator, but details of the molecular mechanism underlying its function remain unclear. Here we show that tubby functions in signal transduction from heterotrimeric GTP-binding protein (G protein)-coupled receptors. Tubby localizes to the plasma membrane by binding phosphatidylinositol 4,5-bisphosphate through its carboxyl terminal "tubby domain." X-ray crystallography reveals the atomic-level basis of this interaction and implicates tubby domains as phosphorylated-phosphatidyl- inositol binding factors. Receptor-mediated activation of G protein alphaq (Galphaq) releases tubby from the plasma membrane through the action of phospholipase C-beta, triggering translocation of tubby to the cell nucleus. The localization of tubby-like protein 3 (TULP3) is similarly regulated. These data suggest that tubby proteins function as membrane-bound transcription regulators that translocate to the nucleus in response to phosphoinositide hydrolysis, providing a direct link between G-protein signaling and the regulation of gene expression.

Two functionally distinct steps mediate high affinity binding of U1A protein to U1 hairpin II RNA.

Binding of the U1A protein to its RNA target U1 hairpin II has been extensively studied as a model for a high affinity RNA/protein interaction. However, the mechanism and kinetics by which this complex is formed remain largely unknown. Here we use real-time biomolecular interaction analysis to dissect the roles various protein and RNA structural elements play in the formation of the U1A.U1 hairpin II complex. We show that neutralization of positive charges on the protein or increasing the salt concentration slows the association rate, suggesting that electrostatic interactions play an important role in bringing RNA and protein together. In contrast, removal of hydrogen bonding or stacking interactions within the RNA/protein interface, or reducing the size of the RNA loop, dramatically destabilizes the complex, as seen by a strong increase in the dissociation rate. Our data support a binding mechanism consisting of a rapid initial association based on electrostatic interactions and a subsequent locking step based on close-range interactions that occur during the induced fit of RNA and protein. Remarkably, these two steps can be clearly distinguished using U1A mutants containing single amino acid substitutions. Our observations explain the extraordinary affinity of U1A for its target and may suggest a general mechanism for high affinity RNA/protein interactions.

Covalent inhibition revealed by the crystal structure of the caspase-8/p35 complex.

Apoptosis is a highly regulated process that is crucial for normal development and homeostasis of multicellular organisms. The p35 protein from baculoviruses effectively prevents apoptosis by its broad-spectrum caspase inhibition. Here we report the crystal structure of p35 in complex with human caspase-8 at 3.0 A resolution, and biochemical and mutagenesis studies based on the structural information. The structure reveals that the caspase is inhibited in the active site through a covalent thioester linkage to p35, which we confirmed by gel electrophoresis, hydroxylamine treatment and mass spectrometry experiments. The p35 protein undergoes dramatic conformational changes on cleavage by the caspase. The repositioning of the amino terminus of p35 into the active site of the caspase eliminates solvent accessibility of the catalytic dyad. This may be crucial for preventing hydrolysis of the thioester intermediate, which is supported by the abrogation of inhibitory activity through mutations at the N terminus of p35. The p35 protein also makes conserved contacts with the caspase outside the active-site region, providing the molecular basis for the broad-spectrum inhibitory activity of this protein. We demonstrate a new molecular mechanism of caspase inhibition, as well as protease inhibition in general.

Structural basis of caspase inhibition by XIAP: differential roles of the linker versus the BIR domain.

The inhibitor of apoptosis proteins (IAPs) represent the only endogenous caspase inhibitors and are characterized by the presence of baculoviral IAP repeats (BIRs). Here, we report the crystal structure of the complex between human caspase-7 and XIAP (BIR2 and the proceeding linker). The structure surprisingly reveals that the linker is the only contacting element for the caspase, while the BIR2 domain is invisible in the crystal. The linker interacts with and blocks the substrate groove of the caspase in a backward fashion, distinct from substrate recognition. Structural analyses suggest that the linker is the energetic and specificity determinant of the interaction. Further biochemical characterizations clearly establish that the linker harbors the major energetic determinant, while the BIR2 domain serves as a regulatory element for caspase binding and Smac neutralization.

Survey of the 1999 surface plasmon resonance biosensor literature.

The application of surface plasmon resonance biosensors in life sciences and pharmaceutical research continues to increase. This review provides a comprehensive list of the commercial 1999 SPR biosensor literature and highlights emerging applications that are of general interest to users of the technology. Given the variability in the quality of published biosensor data, we present some general guidelines to help increase confidence in the results reported from biosensor analyses.

Binding kinetics and ligand specificity for the interactions of the C2B domain of synaptogmin II with inositol polyphosphates and phosphoinositides.

Synaptotagmin II (Syt II) is a key protein in the calcium-dependent exocytosis of synaptic vesicles. It contains two domains homologous to the C2 regulatory region of protein kinase C. The C2A domain acts as a calcium sensor, while the C2B domain has high affinity for inositol polyphosphates (InsP(n)()s) and phosphoinositide polyphosphates (PtdInsP(n)()s). We describe the use of a surface plasmon resonance biosensor in determining the binding kinetics of the C2B domain with InsP(n)() and PtdInsP(n) ligands. Biosensor surfaces were prepared with covalently attached Ins(1,4,5)P(3), Ins(1,3,4,5)P(4), and InsP(6) ligands. The interactions of bacterially expressed His(6)-tagged C2B and (C2A+C2B) domains of Syt II were examined in the presence and absence of competing InsP(n)s and PtdInsP(n)s. Both His(6)-C2B and His(6)-(C2A+C2B) exhibited the highest affinity for the Ins(1,3,4,5)P(4)-modified surface with a K(D) value of 6 nM. The His(6)-(C2A+C2B) had a 10-fold lower association rate constant for the InsP(6)-linked surface (k(a) = 4.6 x 10(3) M(-1) s(-1)) than for the Ins(1,3,4,5)P(4)-modified surface (k(a) = 6.8 x 10(4) M(-1) s(-1)). Two water-soluble phosphoinositides, dioctanoyl-PtdIns(3,4,5)P(3) and dioctanoyl-PtdIns(4,5)P(2), were superior to the soluble InsP(n)s in displacing binding to the Ins(1,3,4,5)P(4)-modified surface. The binding of His(6)-C2B and His(6)-(C2A+C2B) to InsP(n) surfaces did not show significant calcium dependence. These data support a model in which the binding of the C2B domain of Syt II to PtdInsP(n)s is important for the docking and/or fusion of the secretory vesicles to the synaptic plasma membrane.

Energetics of the HIV gp120-CD4 binding reaction.

HIV infection is initiated by the selective interaction between the cellular receptor CD4 and gp120, the external envelope glycoprotein of the virus. We used analytical ultracentrifugation, titration calorimetry, and surface plasmon resonance biosensor analysis to characterize the assembly state, thermodynamics, and kinetics of the CD4-gp120 interaction. The binding thermodynamics were of unexpected magnitude; changes in enthalpy, entropy, and heat capacity greatly exceeded those described for typical protein-protein interactions. These unusual thermodynamic properties were observed with both intact gp120 and a deglycosylated and truncated form of gp120 protein that lacked hypervariable loops V1, V2, and V3 and segments of its N and C termini. Together with previous crystallographic studies, the large changes in heat capacity and entropy reveal that extensive structural rearrangements occur within the core of gp120 upon CD4 binding. CD spectral studies and slow kinetics of binding support this conclusion. These results indicate considerable conformational flexibility within gp120, which may relate to viral mechanisms for triggering infection and disguising conserved receptor-binding sites from the immune system.

A novel mechanism of TRAF signaling revealed by structural and functional analyses of the TRADD-TRAF2 interaction.

TRAF proteins are major mediators for the cell activation, cell survival, and antiapoptotic functions of the TNF receptor superfamily. They can be recruited to activated TNF receptors either by direct interactions with the receptors or indirectly via the adaptor protein TRADD. We now report the structure of the TRADD-TRAF2 complex, which is highly distinct from receptor-TRAF2 interactions. This interaction is significantly stronger and we show by an in vivo signaling assay that TRAF2 signaling is more readily initiated by TRADD than by direct receptor-TRAF2 interactions. TRADD is specific for TRAF1 and TRAF2, which ensures the recruitment of clAPs for the direct inhibition of caspase activation in the signaling complex. The stronger affinity and unique specificity of the TRADD-TRAF2 interaction are crucial for the suppression of apoptosis and provide a mechanistic basis for the perturbation of TRAF recruitment in sensitizing cell death induction.

HuD RNA recognition motifs play distinct roles in the formation of a stable complex with AU-rich RNA.

Human neuron-specific RNA-binding protein HuD belongs to the family of Hu proteins and consists of two N-terminal RNA recognition motifs (RRM1 and -2), a hinge region, and a C-terminal RRM (RRM3). Hu proteins can bind to AU-rich elements in the 3' untranslated regions of unstable mRNAs, causing the stabilization of certain transcripts. We have studied the interaction between HuD and prototype mRNA instability elements of the sequence UU(AUUU)(n)AUU using equilibrium methods and real-time kinetics (surface plasmon resonance using a BIACORE). We show that a single molecule of HuD requires at least three AUUU repeats to bind tightly to the RNA. Deletion of RRM1 reduced the K(d) by 2 orders of magnitude and caused a decrease in the association rate and a strong increase in the dissociation rate of the RNA-protein complex, as expected when a critical RNA-binding domain is removed. In contrast, deletion of either RRM2 or -3, which only moderately reduced the affinity, caused marked increases in the association and dissociation rates. The slower binding and stabilization of the complex observed in the presence of all three RRMs suggest that a change in the tertiary structure occurs during binding. The individual RRMs bind poorly to the RNA (RRM1 binds with micromolar affinity, while the affinities of RRM2 and -3 are in the millimolar range). However, the combination of RRM1 and either RRM2 or RRM3 in the context of the protein allows binding with a nanomolar affinity. Thus, the three RRMs appear to cooperate not only to increase the affinity of the interaction but also to stabilize the formed complex. Kinetic effects, similar to those described here, could play a role in RNA binding by many multi-RRM proteins and may influence the competition between proteins for RNA-binding sites and the ability of RNA-bound proteins to be transported intracellularly.

Surface plasmon resonance kinetic studies of the HIV TAR RNA kissing hairpin complex and its stabilization by 2-thiouridine modification.

Surface plasmon resonance (BIACORE) was used to determine the kinetic values for formation of the HIV TAR-TAR* ('kissing hairpin') RNA complex. The TAR component was also synthesized with the modified nucleoside 2-thiouridine at position 7 in the loop and the kinetics and equilibrium dissociation constants compared with the unmodified TAR hairpin. The BIACORE data show an equilibrium dissociation constant of 1.58 nM for the complex containing the s(2)U modified TAR hairpin, which is 8-fold lower than for the parent hairpin (12.5 nM). This is a result of a 2-fold faster k(a) (4.14x10(5) M(-1) s(-1) versus 2.1x10(5) M(-1) s(-1)) and a 4-fold slower k(d) (6.55x10(-4) s(-1) versus 2.63x10(-3) s(-1)). (1)H NMR imino spectra show that the secondary structure interactions involved in complex formation are retained in the s(2)U-modified complex. Magnesium has been reported to significantly stabilize the TAR-TAR* complex and we found that Mn(2+) and Ca(2+) are also strongly stabilizing, while Mg(2+) exhibited the greatest effect on the complex kinetics. The stabilizing effects of 2-thiouridine indicate that this base modification may be generally useful as an antisense RNA modification for oligonucleotide therapeutics which target RNA loops.