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A correlated motif approach for finding short linear motifs from protein interaction networks.

BACKGROUND: An important class of interaction switches for biological circuits and disease pathways are short binding motifs. However, the biological experiments to find these binding motifs are often laborious and expensive. With the availability of protein interaction data, novel binding motifs can be discovered computationally: by applying standard motif extracting algorithms on protein sequence sets each interacting with either a common protein or a protein group with similar properties. The underlying assumption is that proteins with common interacting partners will share some common binding motifs. Although novel binding motifs have been discovered with such approach, it is not applicable if a protein interacts with very few other proteins or when prior knowledge of protein group is not available or erroneous. Experimental noise in input interaction data can further deteriorate the dismal performance of such approaches. RESULTS: We propose a novel approach of finding correlated short sequence motifs from protein-protein interaction data to effectively circumvent the above-mentioned limitations. Correlated motifs are those motifs that consistently co-occur only in pairs of interacting protein sequences, and could possibly interact with each other directly or indirectly to mediate interactions. We adopted the (l, d)-motif model and formulate finding the correlated motifs as an (l, d)-motif pair finding problem. We present both an exact algorithm, D-MOTIF, as well as its approximation algorithm, D-STAR to solve this problem. Evaluation on extensive simulated data showed that our approach not only eliminated the need for any prior protein grouping, but is also more robust in extracting motifs from noisy interaction data. Application on two biological datasets (SH3 interaction network and TGFbeta signaling network) demonstrates that the approach can extract correlated motifs that correspond to actual interacting subsequences. CONCLUSION: The correlated motif approach outlined in this paper is able to find correlated linear motifs from sparse and noisy interaction data. This, in turn, will expedite the discovery of novel linear binding motifs, and facilitate the studies of biological pathways mediated by them.

Algorithms↗

Significance of aromatic-backbone amide interactions in protein structure.

Weakly polar interactions between aromatic rings of amino acids and hydrogens of backbone amides (Ar-HN) have been shown to support local structures in proteins. Their role in secondary structures, however, has not been elucidated. To investigate the relationship between Ar-HN interaction and the stability of local and secondary structures of polypeptides and to improve the prediction of this interaction based on amino acid sequence, the structures of 560 nonhomologous proteins, from the Protein Data Bank, were searched for Ar-HN interactions between the aromatic ring of each Phe, Tyr, and Trp residue at position i and the backbone amide group of any residue, except Pro, at the positions i, i - 1, i - 2, i - 3, i + 1, i + 2, and i + 3. Ar-HN interactions were identified by calculating the chemical shift of the amide hydrogen caused by the proximal aromatic ring. Ar(i)-HN(i + 1, i + 2 and i + 3) interactions were more common (7.10%, 2.08%, and 0.54%, respectively) than were Ar(i)-HN(i - 1, i - 2, and i - 3) interactions (0.66%, <0.1%, and 0.18%, respectively). The value of the chi(1) torsion angle of the aromatic residue in position i depended on the direction of the Ar-HN interaction. The position of the aromatic ring in Ar(i)-HN(i + 1, i + 2, and i + 3) interactions was mostly trans, in Ar(i)-HN(i - 1, i - 2, and i - 3) interactions mainly gauche(-), and in Ar(i)-HN(i) interactions mostly gauche(+). The analyses of the secondary structures of the protein fragments containing Ar-HN interactions showed that Ar-HN interactions were in all types of secondary structures. Search results suggest that Ar-HN interactions have a stabilizing effect on all types of secondary structures.

Amides↗

Evaluation of the energetic contribution of interhelical Coulombic interactions for coiled coil helix orientation specificity.

Coiled coils are formed by two or more alpha-helices that align in a parallel or an antiparallel relative orientation. The factors that determine a preference for a given relative helix orientation are incompletely understood. The helix orientation preference for the designed coiled coil, Acid-a1-Base-a1, was measured previously. This model system therefore provides a means for the experimental determination of the energetic contribution of a variety of interactions to helix orientation specificity. The antiparallel preference for Acid-a1-Base-a1 is imparted by a single buried polar interaction. Interhelical Coulombic interactions between residues at the e and g positions have been proposed to influence helix orientation preference. In the Acid-a1-Base-a1 heterodimer, potentially attractive Coulombic interactions are expected in both orientations. To determine the energetic consequences of Coulombic interactions for helix orientation preference, we have positioned a single charged residue in each peptide such that exclusively favorable interhelical Coulombic interactions can occur only in the parallel orientation. In contrast, two potentially repulsive interactions are expected in the antiparallel orientation. Because the buried polar interaction can occur only in the antiparallel orientation, interhelical Coulombic interactions favor the parallel orientation and the potential to form a buried polar interaction favors the antiparallel orientation. We find no clear preference for an antiparallel orientation in the resulting heterodimer, Acid-Ke-Base-Eg, suggesting that interhelical Coulombic interactions and a buried polar interaction are of approximately equal importance for helix orientation specificity. Stability measurements indicate that maintenance of all favorable electrostatic interactions and/or avoidance of two potentially repulsive interactions contributes approximately 2.1 kcal/mol to helix orientation preference.

Amino Acid Sequence↗

Bisubstrates: substances that interact with both, renal contraluminal organic anion and organic cation transport systems. II. Zwitterionic substrates: dipeptides, cephalosporins, quinolone-carboxylate gyrase inhibitors and phosphamide thiazine carboxylates; nonionizable substrates: steroid hormones and cyclophosphamides.

In order to test what chemical structure is required for a substrate to interact not only with the contraluminal organic anion (p-aminohippurate, PAH) transporter, but also with the organic cation (N1-methylnicotinamide, NMeN, or tetraethylammonium, TEA) transporter, the stop-flow peritubular capillary perfusion method was applied and app. Ki values were evaluated. Zwitterionic hydrophobic dipeptides not only interact with PAH but also with NMeN transport although with lower inhibitory potency (Ki,PAH = 0.2-1.4; Ki,NMeN 6-14 mmol/l). Amongst the zwitterionic cephalosporins, which all inhibit PAH transport, the amino cephalosporin analogue cefadroxil was identified to interact also with NMeN transport (Ki,PAH = 3.0, Ki,NMeN = 11.2 mmol/l). All zwitterionic naphthyridine and oxochinoline gyrase inhibitors tested inhibit NMeN transport with app. Ki,NMeN values between 1.2 mmol/l and 4.7 mmol/l; the naphthyridine analogues show a good inhibitory potency against PAH transport (Ki,PAH approximately 0.4 mmol/l), the piperazine-containing quinolone analogues have a moderate inhibitory potency (Ki,PAH = 1.1-2.5 mmol/l) and the piperazine-containing pipemidic acid did not inhibit PAH transport at all. Zwitterionic thiazolidine carboxylate phosphamides also interact with both transporters (app. Ki,PAH approximately 3.0; app. Ki,NMeN approximately 18.0 mmol/l). The nonionizable oxo- and hydroxy-group-containing corticosteroid hormones also interact with the two transporters. (a) An OH group in position 21 is necessary for interaction with the PAH transporter, but not for interaction with the TEA transporter. (b) Introduction of an OH group in position 17 alpha abolishes interaction with the TEA transporter, but has different effects with the PAH transporter. (c) Introduction of an OH group in position 6 abolishes interaction with both, the PAH and the TEA transporter. (d) A change of the side-group in position 11 of corticosterone from -OH to -H to = O enhances interaction with the PAH transporter but has no effect on the interaction with the TEA transporter. Nonionizable 4- or 5-androstene analogues inhibit both transporters with app. Ki between 0.16 mmol/l and 0.64 mmol/l, if the steroids are soluble in a concentration greater than 1 mmol/l. Nonionizable oxazaphosphorins with more than one chloroethyl group interact with the PAH transporter with app. Ki between 0.84 mmol/l and 4.9 mmol/l and with the NMeN transporter with app. Ki between 3.2 mmol/l and 18.7 mmol/l. Thus a substrate interacts with both transporters if it is sufficiently hydrophobic, possesses acidic and/or electron-attracting plus basic and/or electron-donating groups, or possesses several electron-attracting nonionizable groups (O, OH, Cl). A certain spatial arrangement of the interacting groups seems to be necessary.

Animals↗

A simple chemical example of hierarchical thermodynamic interactions: the protonation equilibria of inorganic polyprotic acids.

A general method for formulating complex thermodynamic systems in terms of hierarchical interactions has been developed, and has been applied in a previous analysis to hemoglobin oxygen binding data. Polyprotic acids can be considered a simple chemical model of thermodynamic interaction between ligand binding events. To further illustrate the hierarchical interaction approach it is applied to the analysis of the thermodynamic interactions between proton binding events in inorganic polyprotic acids. pK values for arsenate, carbonate, chromate, phosphate, phosphite, selenite, sulfide and sulfite were recast into hierarchical interaction terms. The intrinsic K(d,h) for protonation ranged from 8.8 x 10(-13) (M) for phosphate to 1.3 x 10(-6) (M) for chromate. Pairwise interactions (K(d,hh)) between protonation events ranged from 1.3 x 10(4) for phosphite to 9.4 x 10(5) for carbonate. Third order interactions (K(d,hhh)) were 0.91 and 0.51 for arsenate and phosphate, respectively, values relatively close to the no interaction value of 1. A principle feature of systems described by hierarchical interactions is that higher order interactions, representing more complex interactions, are less likely to be significant than lower order interactions, and this is further illustrated by these observations from polyprotic acids. The set of significant hierarchical interaction values can be used to predict values for as yet unobserved events, and projected pK values are made for all the polyprotic acids included in this study. Finally, application of this method to the protonation equilibria of water demonstrates a profound pairwise interaction between protonation events (K(d,hh) = 1.3 x 10(17)), which is attributed to oxygen's small size and lack of polarizability.

Acids↗

The energetics of ion-pair and hydrogen-bonding interactions in a helical peptide.

A single pair of Glu and Lys residues has been placed at four different spacings, and in both orientations, in an otherwise neutral alanine-glutamine peptide helix, and the contribution to helix stability of the different Glu-Lys interactions has been measured. The contribution from the interaction of each charged side chain with the helix macrodipole has also been determined. A side-chain interaction between Gln and Glu, when the spacing is (i,i+4), has been detected and quantified. The interactions have been divided into contributions from hydrogen bonds (independent of the concentration of NaCl) and from electrostatic interactions (present in 10 mM NaCl, absent in 2.5 M NaCl). The major results are as follows: (1) The (i,i+3) and (i,i+4) Glu-Lys interactions are helix-stabilizing and are similar in strength to each other, regardless of the orientation of the side chains. (2) Hydrogen bonds provide the major contribution to these side-chain interactions, as shown by the following facts. First, the major part of the interaction observed in 10 mM NaCl, pH 7, is still present in 2.5 M NaCl. Second, the interaction found at pH 2 is equally as strong as that found in 2.5 M NaCl at pH 7. (3) The (i,i+4) Gln-Glu side-chain hydrogen bond is as strong as the hydrogen-bond component of the Glu-Lys interaction at both pH 2 and pH 7. The Gln-Glu interaction differs from the Glu-Lys interaction in being specific both for the orientation and the spacing of the residues. (4) No significant hydrogen-bonding interaction was found for the (i,i+1) or (i, i+2) Glu-Lys spacings, either at pH 2 or at pH 7, in 2.5 M NaCl. At 10 mM NaCl and pH 7, these spacings show a helix-destabilizing electrostatic interaction which probably results from stabilization of the coil conformation.

Amino Acid Sequence↗

The contribution of individual interchain interactions to the stabilization of the T and R states of Escherichia coli aspartate transcarbamoylase.

Stabilization of the T and R allosteric states of Escherichia coli aspartate transcarbamoylase is governed by specific intra- and interchain interactions. The six interchain interactions between Glu-239 in one catalytic chain of one catalytic trimer with both Lys-164 and Tyr-165 of a different catalytic chain in the other catalytic trimer have been shown to be involved in the stabilization of the T state. In this study a series of hybrid versions of aspartate transcarbamoylase was studied to determine the minimum number of these Glu-239 interactions necessary to maintain homotropic cooperativity and the T allosteric state. Hybrids with zero, one, and two Glu-239 stabilizing interactions do not exhibit cooperativity, whereas the hybrids with three or more Glu-239 stabilizing interactions exhibit cooperativity. The hybrid enzymes with one or more of the Glu-239 stabilizing interactions also exhibit heterotropic interactions. Two hybrids with three Glu-239 stabilizing interactions, in different geometric relationships, had identical properties. From this and previous studies, it is concluded that the 239 stabilizing interactions play a critical role in the manifestation of homotropic cooperativity in aspartate transcarbamoylase by the stabilization of the T state of the enzyme. As substrate binding energy is utilized, more and more of the T state stabilizing interactions are relaxed, and finally the enzyme shifts to the R state. In the case of the Glu-239 stabilizing interactions more than three of the interactions must be broken before the enzyme shifts to the R state. The interactions between the catalytic and regulatory chains and between the two catalytic trimers of aspartate transcarbamoylase provide a global set of interlocking interactions that stabilize the T and R states of the enzyme. The substrate-induced local conformational changes observed in the structure of the isolated catalytic subunit drive the quaternary T to R transition of aspartate transcarbamoylase and functionally induced homotropic cooperativity.

Adenosine Triphosphate↗

Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

Amino Acid Sequence↗

Protein interactions: two methods for assessment of the reliability of high throughput observations.

High throughput methods for detecting protein interactions require assessment of their accuracy. We present two forms of computational assessment. The first method is the expression profile reliability (EPR) index. The EPR index estimates the biologically relevant fraction of protein interactions detected in a high throughput screen. It does so by comparing the RNA expression profiles for the proteins whose interactions are found in the screen with expression profiles for known interacting and non-interacting pairs of proteins. The second form of assessment is the paralogous verification method (PVM). This method judges an interaction likely if the putatively interacting pair has paralogs that also interact. In contrast to the EPR index, which evaluates datasets of interactions, PVM scores individual interactions. On a test set, PVM identifies correctly 40% of true interactions with a false positive rate of approximately 1%. EPR and PVM were applied to the Database of Interacting Proteins (DIP), a large and diverse collection of protein-protein interactions that contains over 8000 Saccharomyces cerevisiae pairwise protein interactions. Using these two methods, we estimate that approximately 50% of them are reliable, and with the aid of PVM we identify confidently 3003 of them. Web servers for both the PVM and EPR methods are available on the DIP website (dip.doe-mbi.ucla.edu/Services.cgi).

Algorithms↗

Test for interaction between two unlinked loci.

Despite the growing consensus on the importance of testing gene-gene interactions in genetic studies of complex diseases, the effect of gene-gene interactions has often been defined as a deviance from genetic additive effects, which is essentially treated as a residual term in genetic analysis and leads to low power in detecting the presence of interacting effects. To what extent the definition of gene-gene interaction at population level reflects the genes' biochemical or physiological interaction remains a mystery. In this article, we introduce a novel definition and a new measure of gene-gene interaction between two unlinked loci (or genes). We developed a general theory for studying linkage disequilibrium (LD) patterns in disease population under two-locus disease models. The properties of using the LD measure in a disease population as a function of the measure of gene-gene interaction between two unlinked loci were also investigated. We examined how interaction between two loci creates LD in a disease population and showed that the mathematical formulation of the new definition for gene-gene interaction between two loci was similar to that of the LD between two loci. This finding motived us to develop an LD-based statistic to detect gene-gene interaction between two unlinked loci. The null distribution and type I error rates of the LD-based statistic for testing gene-gene interaction were validated using extensive simulation studies. We found that the new test statistic was more powerful than the traditional logistic regression under three two-locus disease models and demonstrated that the power of the test statistic depends on the measure of gene-gene interaction. We also investigated the impact of using tagging SNPs for testing interaction on the power to detect interaction between two unlinked loci. Finally, to evaluate the performance of our new method, we applied the LD-based statistic to two published data sets. Our results showed that the P values of the LD-based statistic were smaller than those obtained by other approaches, including logistic regression models.

Alleles↗

Integrative approach for computationally inferring protein domain interactions.

MOTIVATION: The current need for high-throughput protein interaction detection has resulted in interaction data being generated en masse through such experimental methods as yeast-two-hybrids and protein chips. Such data can be erroneous and they often do not provide adequate functional information for the detected interactions. Therefore, it is useful to develop an in silico approach to further validate and annotate the detected protein interactions. RESULTS: Given that protein-protein interactions involve physical interactions between protein domains, domain-domain interaction information can be useful for validating, annotating, and even predicting protein interactions. However, large-scale, experimentally determined domain-domain interaction data do not exist. Here, we describe an integrative approach to computationally derive putative domain interactions from multiple data sources, including protein interactions, protein complexes, and Rosetta Stone sequences. We further prove the usefulness of such an integrative approach by applying the derived domain interactions to predict and validate protein-protein interactions. AVAILABILITY: A database of putative protein domain interactions derived using the method described in this paper is available at http://interdom.lit.org.sg.

Algorithms↗

Interaction of DNA with clusters of amino acids in proteins.

Protein-DNA interactions facilitate the fundamental functions of living cells and are universal in all living organisms. Several investigations have been carried out, essentially identifying pairs of interactions between the amino acid residues in proteins and the bases in DNA. In the present study, we have detected the recognition motifs that may constitute a cluster of spatially interacting residues in proteins, which interact with the bases of DNA. Graph spectral algorithm has been used to detect side chain clusters comprising Arg, Lys, Asn, Gln and aromatic residues from proteins interacting with DNA. We find that the interaction of proteins with DNA is through clusters in about half of the proteins in the dataset and through individual residues in the rest. Furthermore, inspection of the clusters has revealed additional interactions in a few cases, which have not been reported earlier. The geometry of the interaction between the DNA base and the protein residue is quantified by the distance d and the angle theta. These parameters have been identified for the cation-pi/H-bond stair motif that was reported earlier. Among the Arg, Lys, Asn and Gln residues, the range of (d, theta) values of the interacting Arg clearly falls into the cation-pi and the hydrogen bond interactions of the 'cation-pi/H-bond' stair motif. Analysis of the cluster composition reveals that the Arg residue is predominant than the Lys, Asn and Gln residues. The clusters are classified into Type I and Type II based on the presence or absence of aromatic residues (Phe, Tyr) in them. Residue conservation in these clusters has been examined. Apart from the conserved residues identified previously, a few more residues mainly Phe, Tyr and Arg have also been identified as conserved and interactive with the DNA. Interestingly, a few residues that are parts of interacting clusters and do not interact directly with the DNA have also been conserved. This emphasizes the importance of recognizing the protein side chain cluster motifs interacting with the DNA, which could serve as signatures of protein-DNA recognition in the families of DNA binding proteins.

Amino Acid Motifs↗

Interaction between discrete causes.

The interaction contrast of disease rates (ICDR), a measure of deviation from an additive model of no interaction, is shown always to be zero or slightly negative when the assumptions of no interaction in the sufficient-component discrete causes model hold. Since all physical sources of interaction which one might postulate between discrete causes imply a deviation from this model, the ICDR is a good parameter to screen for causal interaction. Specific models which deviate from the assumptions of no interaction in the sufficient-component discrete causes model should be sought when the ICDR differs from zero. An example of parameterizing interaction in one such model is presented. The additive model is inconsistent with the multiplicative when two causes are studied; negative or no interaction in the multiplicative model might represent positive interaction in the additive model. Use of the multiplicative model to screen for causal interactions could thus lead to inappropriate decisions regarding the need to seek causal explanations for interaction. Since positive interaction in the multiplicative model implies positive interaction in the additive, there will be no such inappropriate decisions when positive interaction is observed in the multiplicative model.

Diabetes Mellitus↗

Hostility, interpersonal interactions, and ambulatory blood pressure.

OBJECTIVE: This study examined aspects of the transactional model of hostility and health by investigating relationships among hostility, interpersonal interactions, and ambulatory blood pressure in a healthy community sample. MATERIALS AND METHODS: Participants included 65 female and 39 male healthy adults between the ages of 18 and 46 years. Ambulatory blood pressure (ABP) and diary data on mood and social interactions were obtained every 20 minutes for 1 day. Mixed models regression analyses were used to evaluate the relationships among hostility, interpersonal interactions, and ABP. RESULTS: Trait hostility was positively associated with the frequency and intensity of negative interactions and was negatively associated with the frequency and intensity of positive interactions. Interacting with others was associated with increases in systolic blood pressure (SBP) and diastolic blood pressure (DBP). The magnitude of the increase in blood pressure was positively associated with the degree to which the interaction was perceived as negative. Hostility was not directly associated with ABP/heart rate (HR) or ABP/HR responses during any interactions or negative interactions. However, there was an interaction between hostility and negative interaction intensity for DBP, suggesting that hostility moderates the effects of negative interactions on DBP. Specifically, increases in the intensity of negative interactions were associated with increases in DBP for participants with high, but not low, hostility. CONCLUSIONS: The results provide partial support for the notion that hostility may be associated with risk for cardiovascular disease through its effects on interpersonal interactions and their cardiovascular correlates.

Adolescent↗

Analysis of verbal interactions in tutorial groups: a process study.

INTRODUCTION: Collaborative learning, including problem-based learning (PBL), is a powerful learning method. Group interaction plays a crucial role in stimulating student learning. However, few studies on learning processes in medical education have examined group interactions. Most studies on collaboration within PBL used self-reported data rather than observational data. We investigated the following types of interactions in PBL tutorial groups: learning-oriented interactions (exploratory questioning, cumulative reasoning and handling conflicts about knowledge); procedural interactions, and irrelevant/off-task interactions. AIM: The central question concerned how much time is spent on the different types of interaction during group sessions and how the types of interaction are distributed over the meeting. METHOD: Four tutorial group sessions in Year 2 of the PBL undergraduate curriculum of Maastricht Medical School were videotaped and analysed. The sessions concerned the reporting phase of the PBL process. We analysed the interactions using a coding scheme distinguishing several verbal interaction types, such as questions, arguments and evaluations. RESULTS: Learning-orientated interactions accounted for 80% of the interactions, with cumulative reasoning, exploratory questioning and handling conflicts about knowledge accounting for about 63%, 10% and 7% of the interactions, respectively. Exploratory questioning often preceded cumulative reasoning. Both types occurred throughout the meeting. Handling conflicts mainly occurred after the first 20 minutes. CONCLUSIONS: Task involvement in the tutorial groups was high. All types of learning-orientated interactions were observed. Relatively little time was spent on exploratory questions and handling conflicts about knowledge. Problem-based learning training should pay special attention to stimulating discussion about contradictory information.

Communication↗

Verbal and social interactions in Swedish forensic psychiatric nursing care as perceived by the patients and nurses.

Patients and nurses in a Swedish forensic psychiatric unit filled in a questionnaire Verbal and Social Interactions designed to survey patients' and nurses' views on the frequency and importance of nursing interactions in forensic psychiatric care. The patients perceived the 'supportive/encouraging interactions' and the 'reality orientation interactions' as the most frequent interactions and the 'supportive/encouraging interactions' and the 'social skills training' as the most important interactions. The nurses perceived the 'supportive/encouraging interactions' and the 'practical skills training' as the most frequent and the 'supportive/encouraging interactions', 'interpretative interactions' and the 'practical skills training' as the most important interactions. There were significant differences between patients' and nurses' perceptions about the frequency of all the different groups of interactions, but greater agreement as to the importance. In general, the patients perceived that the interactions occurred less frequently than the nurses. The differences between patients' and nurses' perceptions on the interactions as well as the clinical implications of these differences are discussed.

Adult↗

Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid protein (pUL86) through two different domains.

We have used the yeast GAL4 two-hybrid system to examine interactions between the human cytomegalovirus (HCMV) major capsid protein (MCP, encoded by UL86) and the precursor assembly protein (pAP, encoded by UL80.5 and cleaved at its carboxyl end to yield AP) and found that (i) the pAP interacts with the MCP through residues located within the carboxy-terminal 21 amino acids of the pAP, called the carboxyl conserved domain (CCD); (ii) the pAP interacts with itself through a separate region, called the amino conserved domain (ACD), located between amino acids His34 and Arg52 near the amino end of the molecule; (iii) the simian CMV (SCMV) pAP and AP can interact with or replace their HCMV counterparts in these interactions, whereas the herpes simplex virus pAP and AP homologs cannot; and (iv) the HCMV and SCMV maturational proteinase precursors (ACpra, encoded by UL80a and APNG1, respectively) can interact with the pAP and MCP. The ACD and CCD amino acid sequences are highly conserved among members of the betaherpesvirus group and appear to have counterparts in the alpha- and gammaherpesvirus pAP homologs. Deleting the ACD from the HCMV pAP, or substituting Ala for a conserved Leu in the ACD, eliminated detectable pAP self-interaction and also substantially reduced MCP binding in the two-hybrid assay. This finding indicates that the pAP self-interaction influences the pAP-MCP interaction. Immunofluorescence studies corroborated the pAP-MCP interaction detected in the GAL4 two-hybrid experiments and showed that nuclear transport of the MCP was mediated by pAP but not AP. We conclude that the pAP interacts with the MCP, that this interaction is mediated by the CCD and is influenced by pAP self-interaction, and that one function of the pAP-MCP interaction may be to provide a controlled mechanism for transporting the MCP into the nucleus.

Amino Acid Sequence↗

Determination of functional domains in the C subunit of the CCAAT-binding factor (CBF) necessary for formation of a CBF-DNA complex: CBF-B interacts simultaneously with both the CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule.

The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and all of which are present in the CBF-DNA complex. In this study using cross-linking and immunoprecipitation methods, we first established that CBF-B interacts simultaneously with both subunits of the CBF-A-CBF-C heterodimer to form a heterotrimeric CBF molecule. We then performed a mutational analysis of CBF-C to define functional interactions with the other two CBF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system. Our experiments established that the evolutionarily conserved segment of CBF-C, which shows similarities with the histone-fold motif of histone H2A, was necessary for formation of the CBF-DNA complex. The domain of CBF-C which interacts with CBF-A included a large portion of this segment, one that corresponds to the segment of the histone-fold motif in H2A used for interaction with H2B. Two classes of interactions involved in formation of the CBF-A-CBF-C heterodimer were detected; one class, provided by residues in the middle of the interaction domain, was needed for formation of the CBF-A-CBF-C heterodimer. The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer. Two separate domains were identified in the conserved segment of CBF-C for interaction with CBF-B; these were located on each side of the CBF-A interaction domain. Since our previous experiments identified a single CBF-B interaction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B. Together with our previous mutational analysis of CBF-A (S. Sinha, I.-S. Kim, K.-Y. Sohn, B. de Crombrugghe, and S. N. Maity, Mol. Cell. Biol. 16:328-337, 1996), this study demonstrates that the histone fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C heterodimer and generate a hybrid surface which then interacts with CBF-B to form the heterotrimeric CBF molecule.

Amino Acid Sequence↗