DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation.

MADS domain proteins are members of a highly conserved family found in all eukaryotes. Genetic studies clearly indicate that many plant MADS domain proteins have different regulatory functions in flower development, yet they share a highly conserved DNA binding domain and can bind to very similar sequences. How, then, can these MADS box genes confer their specific functions? Here, we describe results from DNA binding studies of AGL1 and AGL2 (for AGAMOUS-like), two Arabidopsis MADS domain proteins that are preferentially expressed in flowers. We demonstrate that both proteins are sequence-specific DNA binding proteins and show that each binding consensus has distinct features, suggestion a mechanism for specificity. In addition, we show that the proteins with more similar amino acid sequences have more similar binding sequences. We also found that AGL2 binds to DNA in vitro as a dimer and determined the region of AGL2 that is sufficient for DNA binding and dimerization. Finally, we show that several plant MADS domain proteins can bind to DNA either as homodimers or as heterodimers, suggesting that the number of different regulators could be much greater than the number of MADS box genes.     

Exploring functional roles of multibinding protein interfaces

Cellular processes are highly interconnected and many proteins are shared in different pathways. Some of these shared proteins or protein families may interact with diverse partners using the same interface regions; such multibinding proteins are the subject of our study. The main goal of our study is to attempt to decipher the mechanisms of specific molecular recognition of multiple diverse partners by promiscuous protein regions. To address this, we attempt to analyze the physicochemical properties of multibinding interfaces and highlight the major mechanisms of functional switches realized through multibinding. We find that only 5% of protein families in the structure database have multibinding interfaces, and multibinding interfaces do not show any higher sequence conservation compared with the background interface sites. We highlight several important functional mechanisms utilized by multibinding families. (a) Overlap between different functional pathways can be prevented by the switches involving nearby residues of the same interfacial region. (b) Interfaces can be reused in pathways where the substrate should be passed from one protein to another sequentially. (c) The same protein family can develop different specificities toward different binding partners reusing the same interface; and finally, (d) inhibitors can attach to substrate binding sites as substrate mimicry and thereby prevent substrate binding.     

The Elav-like proteins bind to AU-rich elements and to the poly(A) tail of mRNA.

The Elav-like proteins are specific mRNA binding proteins which are required for cellular differentiation. They contain three characteristic RNP2/RNP1-type RNA binding motifs. Previously we have shown that the first and second RNA binding domains bind to AU-rich elements in the 3'-UTR of mRNA. In this paper we show that the Elav-like proteins exhibit poly(A) binding activity. This activity is distinct from poly(A) binding activities that have been previously described. The Elav-like proteins specifically bind to long chain poly(A) tails. We have shown that the third RNA binding domain encompasses this poly(A) binding activity. Using poly(A)-Sepharose beads in a 'sandwich' assay we have shown that the Elav-like proteins can bind simultaneously to the AU-rich element and to the poly(A) tail.     

Specificity of binding of NH2-terminal residue of proteins to ubiquitin-protein ligase. Use of amino acid derivatives to characterize specific binding sites

Previous studies have indicated that at least part of the selection of proteins for degradation takes place at a binding site on ubiquitin-protein ligase, to which the protein substrate is bound prior to ligation to ubiquitin. It was also shown that proteins with free NH2-terminal alpha-NH2 groups bind better to this site than proteins with blocked NH2 termini (Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999). In the present study, we used simple derivatives of amino acids, such as methyl esters, hydroxamates, or dipeptides, to examine the question of whether the protein binding site of the ligase is able to distinguish between different NH2-terminal residues of proteins. Based on specific patterns of inhibition of the binding to ligase by these derivatives, three types of protein substrates could be distinguished. Type I substrates are proteins that have a basic NH2-terminal residue (such as ribonuclease and lysozyme); these are specifically inhibited by derivatives of the 3 basic amino acids (His, Arg, and Lys) with respect to degradation, ligation to ubiquitin, and binding to ligase. Type II substrates (such as beta-lactoglobulin or pepsinogen, that have a Leu residue at the NH2 terminus) are not affected by the above compounds, but are specifically inhibited by derivatives of bulky hydrophobic amino acids (Leu, Trp, Phe, and Tyr). In these cases, the amino acid derivatives apparently act as specific inhibitors of the binding of the NH2-terminal residue of proteins, as indicated by the following observations: (a) derivatives in which the alpha-NH2 group is blocked were inactive and (b) in dipeptides, the inhibitory amino acid residue had to be at the NH2-terminal position. An additional class (Type III) of substrates comprises proteins that have neither basic nor bulky hydrophobic NH2-terminal amino acid residues; the binding of these proteins is not inhibited by homologous amino acid derivatives that have NH2-terminal residues similar to that of the protein. It is concluded that Type I and Type II proteins bind to distinct and separate subsites of the ligase, specific for basic or bulky hydrophobic NH2-terminal residues, respectively. On the other hand, Type III proteins apparently predominantly interact with the ligase at regions of the protein molecule other than the NH2-terminal residue.     

Structure of yeast hexokinase: IV. Low-resolution structure of enzyme—Substrate complexes revealing negative co-operativity and allosteric interactions

We conclude from X-ray diffraction studies at low resolution (7 Å) that the binding of sugar and nucleotide substrates to dimeric yeast hexokinase BII crystals exhibits both negative co-operativity and positive allosteric co-operativity. Difference electron density maps show the positions of sugar and nucleotide binding sites and extensive substrate-induced structural changes in the protein. Sugar substrates and inhibitors bind in the deep cleft that divides each subunit into two lobes and nucleotide substrates bind nearby to one site per dimer, which lies between the subunits and on the molecular symmetry axis. Although the inhibitors o- and p-iodobenzoylglucosamine and o-toluoylglucosamine bind equally to both subunits, the degree of substitution of glucose or xylose is very different for the two subunits. The substrate analog β, γ-imido ATP shows only one strong binding site per dimer. This negative co-operativity in substrate binding may result from the heterologous or non-equivalent association of the two subunits (Anderson et al., 1974), which provides non-equivalent environments for the two chemically identical subunits.Further, there is a positive allosteric interaction between the sugar and nucleotide binding sites. Sugar binding is required for nucleotide binding at the intersubunit site and the binding of nucleotide modifies the binding of sugars. These positive heterotropic interactions appear to be mediated by extensive substrate-induced structural changes in the enzyme.     

Positive selection and subfunctionalization of duplicated CCT chaperonin subunits

To reach a functional and energetically stable conformation, many proteins need molecular helpers called chaperonins. Among the group II chaperonins, CCT proteins provide crucial machinery for the stabilization and proper folding of several proteins in the cytosol of eukaryotic cells through interactions that are subunit-specific and geometry-dependent. CCT proteins are made up of eight different subunits, all with similar sequences, positioned in a precise arrangement. Each subunit has been proposed to have a specialized function during the binding and folding of the CCT protein substrate. Here, we demonstrate that functional divergence occurred after several CCT duplication events due to the fixation of amino acid substitutions by positive selection. Sites critical for ATP binding and substrate binding were found to have undergone positive selection and functional divergence predominantly in subunits that bind tubulin but not actin. Furthermore, we show clear functional divergence between CCT subunits that bind the C-terminal domains of actin and tubulin and those that bind the N-terminal domains. Phylogenetic analyses could not resolve the deep relationships between most subunits, except for the groups alpha/beta/eta and delta/epsilon, suggesting several almost simultaneous ancient duplication events. Together, the results support the idea that, in contrast to homo-oligomeric chaperonins such as GroEL, the high divergence level between CCT subunits is the result of positive selection after each duplication event to provide a specialized role for each CCT subunit in the different steps of protein folding.     

The lectin chaperone calnexin utilizes polypeptide-based interactions to associate with many of its substrates in vivo

Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum that bind to newly synthesized glycoproteins in part through a lectin site specific for monoglucosylated (Glc(1)Man(7-9)GlcNAc(2)) oligosaccharides. In addition to this lectin-oligosaccharide interaction, in vitro studies have demonstrated that calnexin and calreticulin can bind to polypeptide segments of both glycosylated and nonglycosylated proteins. However, the in vivo relevance of this latter interaction has been questioned. We examined whether polypeptide-based interactions occur between calnexin and its substrates in vivo using the glucosidase inhibitor castanospermine or glucosidase-deficient cells to prevent the formation of monoglucosylated oligosaccharides. We show that if care is taken to preserve weak interactions, the block in lectin-oligosaccharide binding leads to the loss of some calnexin-substrate complexes, but many others remain readily detectable. Furthermore, we demonstrate that calnexin is capable of associating in vivo with a substrate that completely lacks Asn-linked oligosaccharides. The binding of calnexin to proteins that lack monoglucosylated oligosaccharides could not be attributed to nonspecific adsorption nor to its inclusion in protein aggregates. We conclude that both lectin-oligosaccharide and polypeptide-based interactions occur between calnexin and diverse proteins in vivo and that the strength of the latter interaction varies substantially between protein substrates.     

Substrate specificity of the two mitochondrial ornithine carriers can be swapped by single mutation in substrate binding site

Mitochondrial carriers are a large family of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has indicated that these transporters have substrate binding sites in a similar location of the central cavity consisting of three major contact points. Here we have characterized mutations of the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 by carrying out transport assays with a set of different substrates. The different substrate specificities of the two isoforms, which share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Altogether the substrate specificity changes demonstrate that Arg-179 and Glu-180 of contact point II bind the C(α) carboxylate and amino group of the substrates, respectively. Residue Glu-77 of contact point I most likely interacts with the terminal amino group of the substrate side chain. Furthermore, it is likely that all three contact points are involved in the substrate-induced conformational changes required for substrate translocation because Arg-179 is probably connected with Arg-275 of contact point III through Trp-224 by cation-π interactions. Mutations at position 179 also affected the turnover number of the ornithine carrier severely, implying that substrate binding to residue 179 is a rate-limiting step of the catalytic transport cycle. Given that Arg-179 is located in the vicinity of the matrix gate, it is concluded that it is a key residue in the opening of the carrier to the matrix side.     

Negative regulation of cell-cycle progression by RINGO/Speedy E

Cell-cycle transitions are controlled by CDKs (cyclin-dependent kinases), whose activation is usually associated with the binding of cyclins. RINGO/Speedy proteins can also bind to and activate CDKs, although they do not have amino acid sequence homology with cyclins. The RINGO/Speedy family members studied so far positively regulate cell-cycle progression. In the present paper, we report the biochemical and functional characterization of RINGO/Speedy E. We show that RINGO/Speedy E is a functionally distant member of this protein family that negatively affects cell-cycle progression. RINGO/Speedy E overexpression inhibits the meiotic progression in Xenopus oocytes as well as the proliferation of mammalian cells. RINGO/Speedy E can bind to endogenous CDK1 and CDK2 in both cellular systems. However, the RINGO/Speedy E-activated CDKs have different substrate specificity than the CDKs activated by other RINGO/Speedy proteins, which may account for their different effects on the cell cycle. Our results indicate that, although all RINGO/Speedy family members can activate CDKs, they may differently regulate cell-cycle progression.     

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity

We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5′-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex.     

Statistical fluctuations in processes of gene expression regulation: consideration of the problem from point of view of statistical mechanics

The regulation of gene expression is a basic problem of biology. In some cases, the gene activity is regulated by specific binding of regulatory proteins to DNA. In terms of statistical mechanics, this binding is described as the process of adsorption of ligands on the one-dimensional lattice and has a probability nature. As a random physical process, the adsorption of regulatory proteins on DNA introduces a noise to the regulation of gene activity. We derived equations, which make it possible to estimate this noise in the case of the binding of the lac repressor to the operator and showed that these estimates correspond to experimental data. Many ligands are able to bind nonspecifically to DNA. Nonspecific binding is characterized by a lesser equilibrium constant but a greater number of binding sites on the DNA, as compared with specific binding. Relations are presented, which enable one to estimate the probability of the binding of a ligand on a specific site and on nonspecific sites on DNA. The competition between specific and nonspecific binding of regulatory proteins plays a great role in the regulation of gene activity. Similar to the one-dimensional "lattice gas" of particles, ligands adsorbed on DNA produce "one-dimensional" pressure on proteins located at the termini of free regions of DNA. This pressure, an analog of osmotic pressure, may be of importance in processes leading to changes in chromatin structure and activation of gene expression.     

Structural signatures of enzyme binding pockets from order-independent surface alignment: a study of metalloendopeptidase and NAD binding proteins

Detecting similarities between local binding surfaces can facilitate identification of enzyme binding sites and prediction of enzyme functions, and aid in our understanding of enzyme mechanisms. Constructing a template of local surface characteristics for a specific enzyme function or binding activity is a challenging task, as the size and shape of the binding surfaces of a biochemical function often vary. Here we introduce the concept of signature binding pockets, which captures information on preserved and varied atomic positions at multiresolution levels. For proteins with complex enzyme binding and activity, multiple signatures arise naturally in our model, forming a signature basis set that characterizes this class of proteins. Both signatures and signature basis sets can be automatically constructed by a method called SOLAR (Signature Of Local Active Regions). This method is based on a sequence-order-independent alignment of computed binding surface pockets. SOLAR also provides a structure-based multiple sequence fragment alignment to facilitate the interpretation of computed signatures. By studying a family of evolutionarily related proteins, we show that for metzincin metalloendopeptidase, which has a broad spectrum of substrate binding, signature and basis set pockets can be used to discriminate metzincins from other enzymes, to predict the subclass of metzincins functions, and to identify specific binding surfaces. Studying unrelated proteins that have evolved to bind to the same NAD cofactor, we constructed signatures of NAD binding pockets and used them to predict NAD binding proteins and to locate NAD binding pockets. By measuring preservation ratio and location variation, our method can identify residues and atoms that are important for binding affinity and specificity. In both cases, we show that signatures and signature basis set reveal significant biological insight.     

The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions

The reduced coenzyme NADH plays a central role in mitochondrial respiratory metabolism. However, reports on the amount of free NADH in mitochondria are sparse and contradictory. We first determined the emission spectrum of NADH bound to proteins using isothermal titration calorimetry combined with fluorescence spectroscopy. The NADH content of actively respiring mitochondria (from potato tubers [Solanum tuberosum cv Bintje]) in different metabolic states was then measured by spectral decomposition analysis of fluorescence emission spectra. Most of the mitochondrial NADH is bound to proteins, and the amount is low in state 3 (substrate + ADP present) and high in state 2 (only substrate present) and state 4 (substrate + ATP). By contrast, the amount of free NADH is low but relatively constant, even increasing a little in state 3. Using modeling, we show that these results can be explained by a 2.5- to 3-fold weaker average binding of NADH to mitochondrial protein in state 3 compared with state 4. This indicates that there is a specific mechanism for free NADH homeostasis and that the concentration of free NADH in the mitochondrial matrix per se does not play a regulatory role in mitochondrial metabolism. These findings have far-reaching consequences for the interpretation of cellular metabolism.     

Facilitated diffusion of a DNA binding protein on chromatin.

Facilitated diffusion accounts for the rapid rate of association of many bacterial DNA binding proteins with specific DNA sequences in vitro. In this mechanism the proteins bind at random to non-specific sites on the DAN and diffuse (by 'sliding' or 'hopping') along the DNA chain until they arrive at their specific functional sites. We have investigated whether such a mechanism can operate in chromatin by using a bacterial DNA binding protein, Escherichia coli RNA polymerase, that depends on linear diffusion to locate initiation sites on DNA. We have measured the competition between chromatin and its free DNA for the formation of initiation complexes. Only the short linker segments exposed by the removal of histone H1 are available for interaction with the polymerase, but the sparsely distributed promoter sites on the linker DNA of such a polynucleosome chain are located at the same rate as those on DNA. We conclude that the polymerase is free to migrate between the separate linker DNA segments of a polynucleosome chain to reach a promoter site. This chain thus permits the 'hopping' of proteins between neighboring linker segments in their search for a target site on the accessible DNA.     

Structural Mechanism for the Specific Assembly and Activation of the Extracellular Signal Regulated Kinase 5 (ERK5) Module

Mitogen-activated protein kinase (MAPK) activation depends on a linear binding motif found in all MAPK kinases (MKK). In addition, the PB1 (Phox and Bem1) domain of MKK5 is required for extracellular signal regulated kinase 5 (ERK5) activation. We present the crystal structure of ERK5 in complex with an MKK5 construct comprised of the PB1 domain and the linear binding motif. We show that ERK5 has distinct protein-protein interaction surfaces compared with ERK2, which is the closest ERK5 paralog. The two MAPKs have characteristically different physiological functions and their distinct protein-protein interaction surface topography enables them to bind different sets of activators and substrates. Structural and biochemical characterization revealed that the MKK5 PB1 domain cooperates with the MAPK binding linear motif to achieve substrate specific binding, and it also enables co-recruitment of the upstream activating enzyme and the downstream substrate into one signaling competent complex. Studies on present day MAPKs and MKKs hint on the way protein kinase networks may evolve. In particular, they suggest how paralogous enzymes with similar catalytic properties could acquire novel signaling roles by merely changing the way they make physical links to other proteins.     

CLIP: a method for identifying protein-RNA interaction sites in living cells

Nucleic-acid binding proteins constitute nearly one-fourth of all functionally annotated human genes. Genome-wide analysis of protein-nucleic acid contacts has not yet been performed for most of these proteins, restricting attempts to establish a comprehensive understanding of protein function. UV cross-linking is a method typically used to determine the position of direct interactions between proteins and nucleic acids. We have developed the cross-linking and immunoprecipitation assay, which exploits the covalent protein-nucleic acid cross-linking to stringently purify a specific protein-RNA complex using immunoprecipitation followed by SDS-PAGE separation. In this way, the vast majority of non-specific contaminating RNA, which can bind to co-immunoprecipitated proteins or beads, can be removed. Here, we present an improved protocol that performs RNA linker ligation before the SDS-PAGE step, and describe its application to the specific purification and amplification of RNA ligands of Nova in neurons.     

Multiple diverse ligands binding at a single protein site: A matter of pre-existing populations

Here, we comment on the steadily increasing body of data showing that proteins with specificity actually bind ligands of diverse shapes, sizes, and composition. Such a phenomenon is not surprising when one considers that binding is a dynamic process with populations in equilibrium and that the shape of the binding site is strongly influenced by the molecular partner. It derives implicitly from the concept of populations. All proteins, specific and nonspecific, exist in ensembles of substates. If the library of ligands in solution is large enough, favorably matching ligands with altered shapes and sizes can be expected to bind, with a redistribution of the protein populations. Point mutations at spatially distant sites may exert large conformational rearrangements and hinge effects, consistent with mutations away from the binding site leading to population shifts and (cross-)drug resistance. A similar effect is observed in protein superfamilies, in which different sequences with similar topologies display similar large-scale dynamic motions. The hinges are frequently at analogous sites, yet with different substrate specificity. Similar topologies yield similar conformational isomers, although with different distributions of population times, owing to the change in the conditions, that is, the change in the sequences. In turn, different distributions relate to binding of different sizes and shapes. Hence, the binding site shape and size are defined by the ligand. They are not independent entities of fixed proportions and cannot be analyzed independently of the binding partner. Such a proposition derives from viewing proteins as dynamic distributions, presenting to the incoming ligands a range of binding site shapes. It illustrates how presumably specific binding molecules can bind multiple ligands. In terms of drug design, the ability of a single receptor to recognize many dissimilar ligands shows the need to consider more diverse molecules. It provides a rationale for higher affinity inhibitors that are not derived from substrates at their transition states and indicates flexible docking schemes.     

Protein scaffolds can enhance the bistability of multisite phosphorylation systems

The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large K(M), compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites.     

The logical repertoire of ligand-binding proteins

Proteins whose conformation can be altered by the equilibrium binding of a regulatory ligand are one of the main building blocks of signal-processing networks in cells. Typically, such proteins switch between an 'inactive' and an 'active' state, as the concentration of the regulator varies. We investigate the properties of proteins that can bind two different ligands and show that these proteins can individually act as logical elements: their 'output', quantified by their average level of activity, depends on the two 'inputs', the concentrations of both regulators. In the case where the two ligands can bind simultaneously, we show that all of the elementary logical functions can be implemented by appropriate tuning of the ligand-binding energies. If the ligands bind exclusively, the logical repertoire is more limited. When such proteins cluster together, cooperative interactions can greatly enhance the sharpness of the response. Protein clusters can therefore act as digital logical elements whose activity can be abruptly switched from fully inactive to fully active, as the concentrations of the regulators pass threshold values. We discuss a particular instance in which this type of protein logic appears to be used in signal transduction-the chemotaxis receptors of E. coli.