scholarly journals Cooperative Binding of KaiB to the KaiC Hexamer Ensures Accurate Circadian Clock Oscillation in Cyanobacteria

2019 ◽  
Vol 20 (18) ◽  
pp. 4550 ◽  
Author(s):  
Reiko Murakami ◽  
Yasuhiro Yunoki ◽  
Kentaro Ishii ◽  
Kazuki Terauchi ◽  
Susumu Uchiyama ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Structural Data ◽  
Cooperative Interaction ◽  
Cooperative Binding ◽  
Rhythm Generation ◽  
Interaction Sites ◽  
Central Oscillator ◽  
Cooperative Assembly ◽  
Distal Mutation ◽  
Clock Protein

The central oscillator generating cyanobacterial circadian rhythms comprises KaiA, KaiB, and KaiC proteins. Their interactions cause KaiC phosphorylation and dephosphorylation cycles over approximately 24 h. KaiB interacts with phosphorylated KaiC in competition with SasA, an output protein harboring a KaiB-homologous domain. Structural data have identified KaiB–KaiC interaction sites; however, KaiB mutations distal from the binding surfaces can impair KaiB–KaiC interaction and the circadian rhythm. Reportedly, KaiB and KaiC exclusively form a complex in a 6:6 stoichiometry, indicating that KaiB–KaiC hexamer binding shows strong positive cooperativity. Here, mutational analysis was used to investigate the functional significance of this cooperative interaction. Results demonstrate that electrostatic complementarity between KaiB protomers promotes their cooperative assembly, which is indispensable for accurate rhythm generation. SasA does not exhibit such electrostatic complementarity and noncooperatively binds to KaiC. Thus, the findings explain KaiB distal mutation effects, providing mechanistic insights into clock protein interplay.

The Role of β-TrCP1 and β-TrCP2 in Circadian Rhythm Generation by Mediating Degradation of Clock Protein PER2

2008 ◽  
Vol 144 (5) ◽  
pp. 609-618 ◽  
Author(s):  
Kanae Ohsaki ◽  
Katsutaka Oishi ◽  
Yuko Kozono ◽  
Keiko Nakayama ◽  
Keiichi I. Nakayama ◽  
...  
Keyword(s):  
Circadian Rhythm ◽  
Rhythm Generation ◽  
Clock Protein

scholarly journals Anti-cooperative assembly of the SRP19 and SRP68/72 components of the signal recognition particle

2008 ◽  
Vol 415 (3) ◽  
pp. 429-437 ◽  
Author(s):  
Tuhin Subhra Maity ◽  
Howard M. Fried ◽  
Kevin M. Weeks
Keyword(s):  
Electrostatic Interactions ◽  
Signal Recognition Particle ◽  
Cooperative Binding ◽  
Signal Recognition ◽  
Parallel Orientation ◽  
Domain Interactions ◽  
Cooperative Assembly ◽  
Srp Rna ◽  
Antagonistic Interactions

The mammalian SRP (signal recognition particle) represents an important model for the assembly and role of inter-domain interactions in complex RNPs (ribonucleoproteins). In the present study we analysed the interdependent interactions between the SRP19, SRP68 and SRP72 proteins and the SRP RNA. SRP72 binds the SRP RNA largely via non-specific electrostatic interactions and enhances the affinity of SRP68 for the RNA. SRP19 and SRP68 both bind directly and specifically to the same two RNA helices, but on opposite faces and at opposite ends. SRP19 binds at the apices of helices 6 and 8, whereas the SRP68/72 heterodimer binds at the three-way junction involving RNA helices 5, 6 and 8. Even though both SRP19 and SRP68/72 stabilize a similar parallel orientation for RNA helices 6 and 8, these two proteins bind to the RNA with moderate anti-cooperativity. Long-range anti-cooperative binding by SRP19 and SRP68/72 appears to arise from stabilization of distinct conformations in the stiff intervening RNA scaffold. Assembly of large RNPs is generally thought to involve either co-operative or energetically neutral interactions among components. By contrast, our findings emphasize that antagonistic interactions can play significant roles in assembly of multi-subunit RNPs.

scholarly journals Fourteen Residues of the U1 snRNP-Specific U1A Protein Are Required for Homodimerization, Cooperative RNA Binding, and Inhibition of Polyadenylation

2000 ◽  
Vol 20 (6) ◽  
pp. 2209-2217 ◽  
Author(s):  
Jacqueline M. T. Klein Gunnewiek ◽  
Reem I. Hussein ◽  
Yvonne van Aarssen ◽  
Daphne Palacios ◽  
Rob de Jong ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Structural Data ◽  
Protein Molecule ◽  
Cooperative Binding ◽  
Mobility Shift ◽  
Small Nuclear Ribonucleoprotein ◽  
U1a Protein

ABSTRACT It was previously shown that the human U1A protein, one of three U1 small nuclear ribonucleoprotein-specific proteins, autoregulates its own production by binding to and inhibiting the polyadenylation of its own pre-mRNA. The U1A autoregulatory complex requires two molecules of U1A protein to cooperatively bind a 50-nucleotide polyadenylation-inhibitory element (PIE) RNA located in the U1A 3′ untranslated region. Based on both biochemical and nuclear magnetic resonance structural data, it was predicted that protein-protein interactions between the N-terminal regions (amino acids [aa] 1 to 115) of the two U1A proteins would form the basis for cooperative binding to PIE RNA and for inhibition of polyadenylation. In this study, we not only experimentally confirmed these predictions but discovered some unexpected features of how the U1A autoregulatory complex functions. We found that the U1A protein homodimerizes in the yeast two-hybrid system even when its ability to bind RNA is incapacitated. U1A dimerization requires two separate regions, both located in the N-terminal 115 residues. Using both coselection and gel mobility shift assays, U1A dimerization was also observed in vitro and found to depend on the same two regions that were found in vivo. Mutation of the second homodimerization region (aa 103 to 115) also resulted in loss of inhibition of polyadenylation and loss of cooperative binding of two U1A protein molecules to PIE RNA. This same mutation had no effect on the binding of one U1A protein molecule to PIE RNA. A peptide containing two copies of aa 103 to 115 is a potent inhibitor of polyadenylation. Based on these data, a model of the U1A autoregulatory complex is presented.

scholarly journals Struct2Graph: A graph attention network for structure based predictions of protein-protein interactions

2020 ◽  
Author(s):  
Mayank Baranwal ◽  
Abram Magner ◽  
Jacob Saldinger ◽  
Emine S. Turali-Emre ◽  
Shivani Kozarekar ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Intracellular Signaling ◽  
Metabolic Regulation ◽  
Selection Process ◽  
3D Structure ◽  
Structural Data ◽  
Sequence Information ◽  
Protein Protein Interactions ◽  
Attention Network ◽  
Interaction Sites

AbstractDevelopment of new methods for analysis of protein-protein interactions (PPIs) at molecular and nanometer scales gives insights into intracellular signaling pathways and will improve understanding of protein functions, as well as other nanoscale structures of biological and abiological origins. Recent advances in computational tools, particularly the ones involving modern deep learning algorithms, have been shown to complement experimental approaches for describing and rationalizing PPIs. However, most of the existing works on PPI predictions use protein-sequence information, and thus have difficulties in accounting for the three-dimensional organization of the protein chains. In this study, we address this problem and describe a PPI analysis method based on a graph attention network, named Struct2Graph, for identifying PPIs directly from the structural data of folded protein globules. Our method is capable of predicting the PPI with an accuracy of 98.89% on the balanced set consisting of an equal number of positive and negative pairs. On the unbalanced set with the ratio of 1:10 between positive and negative pairs, Struct2Graph achieves a five-fold cross validation average accuracy of 99.42%. Moreover, unsupervised prediction of the interaction sites by Struct2Graph for phenol-soluble modulins are found to be in concordance with the previously reported binding sites for this family.Author summaryPPIs are the central part of signal transduction, metabolic regulation, environmental sensing, and cellular organization. Despite their success, most strategies to decode PPIs use sequence based approaches do not generalize to broader classes of chemical compounds of similar scale as proteins that are equally capable of forming complexes with proteins that are not based on amino acids, and thus lack of an equivalent sequence-based representation. Here, we address the problem of prediction of PPIs using a first of its kind, 3D structure based graph attention network (available at https://github.com/baranwa2/Struct2Graph). Despite its excellent prediction performance, the novel mutual attention mechanism provides insights into likely interaction sites through its knowledge selection process in a completely unsupervised manner.

scholarly journals Mutations in Sensor 1 and Walker B in the Bovine Papillomavirus E1 Initiator Protein Mimic the Nucleotide-Bound State

2009 ◽  
Vol 84 (4) ◽  
pp. 1912-1919 ◽  
Author(s):  
Xiaofei Liu ◽  
Arne Stenlund
Keyword(s):  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Structural Data ◽  
Bound State ◽  
Atp Binding ◽  
Bovine Papillomavirus ◽  
Viral Genomes ◽  
Initiator Protein ◽  
Close Proximity ◽  
Atp Binding And Hydrolysis

ABSTRACT Viral replication initiator proteins are multifunctional proteins that utilize ATP binding and hydrolysis by their AAA+ modules for multiple functions in the replication of their viral genomes. These proteins are therefore of particular interest for understanding how AAA+ proteins carry out multiple ATP driven functions. We have performed a comprehensive mutational analysis of the residues involved in ATP binding and hydrolysis in the papillomavirus E1 initiator protein based on the recent structural data. Ten of the eleven residues that were targeted were defective for ATP hydrolysis, and seven of these were also defective for ATP binding. The three mutants that could still bind nucleotide represent the Walker B motif (D478 and D479) and Sensor 1 (N523), three residues that are in close proximity to each other and generally are considered to be involved in ATP hydrolysis. Surprisingly, however, two of these mutants, D478A and N523A, mimicked the nucleotide bound state and were capable of binding DNA in the absence of nucleotide. However, these mutants could not form the E1 double trimer in the absence of nucleotide, demonstrating that there are two qualitatively different consequences of ATP binding by E1, one that can be mimicked by D478A and N523A and one which cannot.

scholarly journals Functional Analysis of the Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase (CobT) Enzyme, Involved in the Late Steps of Coenzyme B12 Biosynthesis in Salmonella enterica

2009 ◽  
Vol 192 (1) ◽  
pp. 145-154 ◽  
Author(s):  
Kathy R. Claas ◽  
J. R. Parrish ◽  
L. A. Maggio-Hall ◽  
J. C. Escalante-Semerena
Keyword(s):  
Salmonella Enterica ◽  
Mutational Analysis ◽  
Residual Activity ◽  
Catalytic Efficiency ◽  
Structural Data ◽  
Site Directed Mutagenesis ◽  
Negative Effects ◽  
Intragenic Complementation

ABSTRACT In Salmonella enterica, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M cobT mutations were deleterious, we determined the nature of three class M cobT alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobTWT) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobTWT. The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M cobT alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.

scholarly journals Identification of a ligand-binding site in an immunoglobulin fold domain of the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

1996 ◽  
Vol 7 (1) ◽  
pp. 143-153 ◽  
Author(s):  
H de Nobel ◽  
P N Lipke ◽  
J Kurjan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ligand Binding ◽  
Binding Site ◽  
Mutational Analysis ◽  
Homology Model ◽  
Ligand Binding Site ◽  
Adhesion Protein ◽  
Interaction Sites ◽  
Critical Residue ◽  
Immunoglobulin Fold

The Saccharomyces cerevisiae adhesion protein alpha-agglutinin (Ag alpha 1p) is expressed by alpha cells and binds to the complementary a-agglutinin expressed by a cells. The N-terminal half of alpha-agglutinin is sufficient for ligand binding and has been proposed to contain an immunoglobulin (Ig) fold domain. Based on a structural homology model for this domain and a previously identified critical residue (His292), we made Ag alpha 1p mutations in three discontinuous patches of the domain that are predicted to be in close proximity to His292 in the model. Residues in each of the three patches were identified that are important for activity and therefore define a putative ligand binding site, whereas mutations in distant loops had no effect on activity. This putative binding site is on a different surface of the Ig fold than the defined binding sites of immunoglobulins and other members of the Ig superfamily. Comparison of protein interaction sites by structural and mutational analysis has indicated that the area of surface contact is larger than the functional binding site identified by mutagenesis. The putative alpha-agglutinin binding site is therefore likely to identify residues that contribute to the functional binding site within a larger area that contacts a-agglutinin.

scholarly journals Structural Determinants of Cholesterol Recognition in Helical Integral Membrane Proteins

2020 ◽  
Author(s):  
B. Marlow ◽  
G. Kuenze ◽  
B. Li ◽  
C. Sanders ◽  
J. Meiler
Keyword(s):  
Molecular Mechanisms ◽  
Search Algorithm ◽  
Three Dimensional ◽  
Structural Data ◽  
Spatial Arrangement ◽  
Integral Membrane Protein ◽  
Membrane Dynamics ◽  
Structural Determinants ◽  
Biologically Relevant ◽  
Interaction Sites

ABSTRACTCholesterol (CLR) is an integral component of mammalian membranes. It has been shown to modulate membrane dynamics and alter integral membrane protein (IMP) function. However, understanding the molecular mechanisms of these processes is complicated by limited and conflicting structural data: Specifically, in co-crystal structures of CLR-IMP complexes it is difficult to distinguish specific and biologically relevant CLR-IMP interactions from a nonspecific association captured by the crystallization process. The only widely recognized search algorithm for CLR-IMP interaction sites is sequence-based, i.e. searching for the so-called ‘CRAC’ or ‘CARC’ motifs. While these motifs are present in numerous IMPs, there is inconclusive evidence to support their necessity or sufficiency for CLR binding. Here we leverage the increasing number of experimental CLR-IMP structures to systematically analyze putative interaction sites based on their spatial arrangement and evolutionary conservation. From this analysis we create three-dimensional representations of general CLR interaction sites that form clusters across multiple IMP classes and classify them as being either specific or nonspecific. Information gleaned from our characterization will eventually enable a structure-based approach for prediction and design of CLR-IMP interaction sites.SIGNIFICANCECLR plays an important role in composition and function of membranes and often surrounds and interacts with IMPs. It is a daunting challenge to disentangle CLRs dual roles as a direct modulator of IMP function through binding or indirect actor as a modulator of membrane plasticity. Only recently studies have delved into characterizing specific CLR-IMP interactions. We build on this previous work by using a combination of structural and evolutionary characteristics to distinguish specific from nonspecific CLR interaction sites. Understanding how CLR interacts with IMPs will underpin future development towards detecting and engineering CLR-IMP interaction sites.

scholarly journals Interactions of the Integrase Protein of the Conjugative Transposon Tn916 with Its Specific DNA Binding Sites

1999 ◽  
Vol 181 (19) ◽  
pp. 6114-6123 ◽  
Author(s):  
Yunhua Jia ◽  
Gordon Churchward
Keyword(s):  
Dna Binding ◽  
Chimeric Protein ◽  
Cooperative Interaction ◽  
Hill Coefficient ◽  
Cooperative Binding ◽  
Mobility Shift ◽  
Dna Molecule ◽  
Terminal Domain ◽  
Electrophoretic Mobilities ◽  
Single Dna Molecule

ABSTRACT The binding of two chimeric proteins, consisting of the N-terminal or C-terminal DNA binding domain of Tn916 Int fused to maltose binding protein, to specific oligonucleotide substrates was analyzed by gel mobility shift assay. The chimeric protein with the N-terminal domain formed two complexes of different electrophoretic mobilities. The faster-moving complex, whose formation displayed no cooperativity, contained two protein monomers bound to a single DNA molecule. The slower-moving complex, whose formation involved cooperative binding (Hill coefficient > 1.0), contained four protein monomers bound to a single DNA molecule. Methylation interference experiments coupled with the analysis of protein binding to mutant oligonucleotide substrates showed that formation of the faster-moving complex containing two protein monomers required the presence of two 11-bp direct repeats (called DR2) in direct orientation. Formation of the slower-moving complex required only a single DR2 repeat. Binding of the N-terminal domains in vivo could serve to position two Int monomers on the DNA near each end of the transposon and assist in bringing together the ends of the transposon so that excision can occur. The chimeric protein with the C-terminal domain of Int also formed two complexes of different electrophoretic mobilities. The major, slower-moving complex, whose formation involved cooperative binding, contained two protein molecules bound to one DNA molecule. This finding suggested that while the C-terminal domain of Int can bind DNA as a monomer, a cooperative interaction between two monomers of the C-terminal domain may help to bring the ends of the transposon together during excision.

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