scholarly journals Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA, EsxB, and EspA

2009 ◽  
Vol 192 (1) ◽  
pp. 326-335 ◽  
Author(s):  
Brian Callahan ◽  
Kiet Nguyen ◽  
Alissa Collins ◽  
Kayla Valdes ◽  
Michael Caplow ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Helical Structure ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Structures And Properties ◽  
Virulence Protein ◽  
Single Polypeptide ◽  
Specific Association

ABSTRACT Mycobacterium tuberculosis EsxA and EsxB proteins are founding members of the WXG100 (WXG) protein family, characterized by their small size (∼100 amino acids) and conserved WXG amino acid motif. M. tuberculosis contains 11 tandem pairs of WXG genes; each gene pair is thought to be coexpressed to form a heterodimer. The precise role of these proteins in the biology of M. tuberculosis is unknown, but several of the heterodimers are secreted, which is important for virulence. However, WXG proteins are not simply virulence factors, since nonpathogenic mycobacteria also express and secrete these proteins. Here we show that three WXG heterodimers have structures and properties similar to those of the M. tuberculosis EsxBA (MtbEsxBA) heterodimer, regardless of their host species and apparent biological function. Biophysical studies indicate that the WXG proteins from M. tuberculosis (EsxG and EsxH), Mycobacterium smegmatis (EsxA and EsxB), and Corynebacterium diphtheriae (EsxA and EsxB) are heterodimers and fold into a predominately α-helical structure. An in vivo protein-protein interaction assay was modified to identify proteins that interact specifically with the native WXG100 heterodimer. MtbEsxA and MtbEsxB were fused into a single polypeptide, MtbEsxBA, to create a biomimetic bait for the native heterodimer. The MtbEsxBA bait showed specific association with several esx-1-encoded proteins and EspA, a virulence protein secreted by ESX-1. The MtbEsxBA fusion peptide was also utilized to identify residues in both EsxA and EsxB that are important for establishing protein interactions with Rv3871 and EspA. Together, the results are consistent with a model in which WXG proteins perform similar biological roles in virulent and nonvirulent species.

scholarly journals Evolutionary diversification of protein–protein interactions by interface add-ons

2017 ◽  
Vol 114 (40) ◽  
pp. E8333-E8342 ◽  
Author(s):  
Maximilian G. Plach ◽  
Florian Semmelmann ◽  
Florian Busch ◽  
Markus Busch ◽  
Leonhard Heizinger ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Evolutionary Strategy ◽  
Structural Level ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Evolutionary Diversification

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein–protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein–protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein–protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein–protein interactions.

scholarly journals A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

1995 ◽  
Vol 15 (10) ◽  
pp. 5214-5225 ◽  
Author(s):  
A D Catling ◽  
H J Schaeffer ◽  
C W Reuter ◽  
G R Reddy ◽  
M J Weber
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Serum Stimulation ◽  
And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

scholarly journals A Conditional Role of U2AF in Splicing of Introns with Unconventional Polypyrimidine Tracts

2007 ◽  
Vol 27 (20) ◽  
pp. 7334-7344 ◽  
Author(s):  
Vinod Sridharan ◽  
Ravinder Singh
Keyword(s):  
Protein Interactions ◽  
Splice Site ◽  
Large Subunit ◽  
Strong Dependence ◽  
Rna Recognition Motif ◽  
Branch Points ◽  
Protein Protein Interactions ◽  
Site Identification

ABSTRACT Recognition of polypyrimidine (Py) tracts typically present between the branch point and the 3′ splice site by the large subunit of the essential splicing factor U2AF is a key early step in pre-mRNA splicing. Diverse intronic sequence arrangements exist, however, including 3′ splice sites lacking recognizable Py tracts, which raises the question of how general the requirement for U2AF is for various intron architectures. Our analysis of fission yeast introns in vivo has unexpectedly revealed that whereas introns lacking Py tracts altogether remain dependent on both subunits of U2AF, introns with long Py tracts, unconventionally positioned upstream of branch points, are unaffected by U2AF inactivation. Nevertheless, mutation of these Py tracts causes strong dependence on the large subunit U2AF59. We also find that Py tract diversity influences the requirement for the conserved C-terminal domain of U2AF59 (RNA recognition motif 3), which has been implicated in protein-protein interactions with other splicing factors. Together, these results suggest that in addition to Py tract binding by U2AF, supplementary mechanisms of U2AF recruitment and 3′ splice site identification exist to accommodate diverse intron architectures, which have gone unappreciated in biochemical studies of model pre-mRNAs.

scholarly journals Identification of protein-protein and ribonucleoprotein complexes containing Hfq

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joël Caillet ◽  
Bruno Baron ◽  
Irina V. Boni ◽  
Célia Caillet-Saguy ◽  
Eliane Hajnsdorf
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Direct Interaction ◽  
Rnase A ◽  
Rnase E ◽  
Protein Protein Interactions ◽  
Control Of Gene Expression ◽  
Ribonucleoprotein Complexes

Abstract Hfq is a RNA-binding protein that plays a pivotal role in the control of gene expression in bacteria by stabilizing sRNAs and facilitating their pairing with multiple target mRNAs. It has already been shown that Hfq, directly or indirectly, interacts with many proteins: RNase E, Rho, poly(A)polymerase, RNA polymerase… In order to detect more Hfq-related protein-protein interactions we have used two approaches, TAP-tag combined with RNase A treatment to access the role of RNA in these complexes, and protein-protein crosslinking, which freezes protein-protein complexes formed in vivo. In addition, we have performed microscale thermophoresis to evaluate the role of RNA in some of the complexes detected and used far-western blotting to confirm some protein-protein interactions. Taken together, the results show unambiguously a direct interaction between Hfq and EF-Tu. However a very large number of the interactions of proteins with Hfq in E. coli involve RNAs. These RNAs together with the interacting protein, may play an active role in the formation of Hfq-containing complexes with previously unforeseen implications for the riboregulatory functions of Hfq.

scholarly journals In silico analysis of protein-protein interfaces: Tools and approaches

2020 ◽  
Vol 11 (4) ◽  
pp. 7539-7548
Author(s):  
Christina Nilofer ◽  
Arumugam Mohanapriya
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
In Silico Analysis ◽  
Physicochemical Characteristics ◽  
Structural Features ◽  
Interface Residue ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Molecular Functions

Two or more proteins interact in vivo to perform complex molecular functions including catalysis, regulation, assembly, immunity and inhibition through the formation of stable interfaces. This interaction is governed by several factors that are selective, sensitive and specific in nature. Several interface features has been documented since 1975. The study of these interface features of proteins and their dynamicity during interaction with different proteins help understanding the mechanisms underlying diverse molecular functions and its biological processes. Computational tools greatly assist in studying such interface features that determine the interaction between two or more proteins, and in this context, this review enumerates the different interface features reported thus far along with the tools that aid in deciphering protein features (physicochemical characteristics, binding site and interface residue prediction and hotspot residues) along with their approaches that are employed in the prediction these features. Also, the review discusses the advantages and limitations of experimental techniques and computational biological tools deployed for deciphering the protein-protein interactions. Altogether, the review will provide insights into the optimal tools and different strategies involved in protein interaction studies that would facilitate the researchers to understand the protein structural features and molecular principles of protein-protein interaction with known functions.

scholarly journals Biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Isseki Yu ◽  
Takaharu Mori ◽  
Tadashi Ando ◽  
Ryuhei Harada ◽  
Jaewoon Jung ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Structures ◽  
Biological Macromolecules ◽  
Atomistic Model ◽  
Protein Protein Interactions ◽  
Structure And Dynamics ◽  
Specific Association ◽  
Dynamics Simulations

Biological macromolecules function in highly crowded cellular environments. The structure and dynamics of proteins and nucleic acids are well characterized in vitro, but in vivo crowding effects remain unclear. Using molecular dynamics simulations of a comprehensive atomistic model cytoplasm we found that protein-protein interactions may destabilize native protein structures, whereas metabolite interactions may induce more compact states due to electrostatic screening. Protein-protein interactions also resulted in significant variations in reduced macromolecular diffusion under crowded conditions, while metabolites exhibited significant two-dimensional surface diffusion and altered protein-ligand binding that may reduce the effective concentration of metabolites and ligands in vivo. Metabolic enzymes showed weak non-specific association in cellular environments attributed to solvation and entropic effects. These effects are expected to have broad implications for the in vivo functioning of biomolecules. This work is a first step towards physically realistic in silico whole-cell models that connect molecular with cellular biology.

scholarly journals CeLINC, a fluorescence-based protein-protein interaction assay in C. elegans

2021 ◽  
Author(s):  
Jason R Kroll ◽  
Sanne Remmelzwaal ◽  
Mike Boxem
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Protein Protein Interactions ◽  
Bait Protein ◽  
C Elegans ◽  
Protein Protein Interaction ◽  
Prey Protein ◽  
Reference Protein

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein's function. We present C. elegans light-induced co-clustering (CeLINC), an optical binary protein-protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein-protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Co-localization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.

scholarly journals Selective secretion of alternatively spliced fibronectin variants.

1989 ◽  
Vol 109 (6) ◽  
pp. 3445-3453 ◽  
Author(s):  
J E Schwarzbauer ◽  
C S Spencer ◽  
C L Wilson
Keyword(s):  
Protein Interactions ◽  
Time Course ◽  
Deletion Mapping ◽  
Protein Protein Interactions ◽  
Dimer Formation ◽  
Alternatively Spliced ◽  
Selective Retention ◽  
V Region

We demonstrate that the alternatively spliced variable (V) region of fibronectin (FN) is required for secretion of FN dimers during biosynthesis. Alternative splicing of the V segment of the rat FN transcript generates three subunit variants (V120, V95, V0) that differ by the inclusion or omission of an additional 120 or 95 amino acids. We are exploring the functions of this segment by expressing variant cDNAs in normal and transformed fibroblasts. Like FN itself, the cDNA-encoded polypeptides (deminectins [DNs]) containing the V120 or V95 segment are efficiently secreted as disulfide-bonded homodimers. However, few homodimers of DNs lacking this region, V0 DNs, are secreted. V0 homodimers do form inside the cell, as demonstrated by biosynthetic analyses of dimer formation and secretion using pulse-chase and time course experiments, but these dimers seldom reach the cell surface and are probably degraded intracellularly. Coexpression of V0 and V120 subunits results in intracellular formation of three types of dimers, V0-V0, V0-V120, and V120-V120, but only the V120-containing dimers are secreted. This selective retention of V0 homodimers indicates that the V region is required for formation and secretion of native FN dimers. In an analogous in vivo situation, we show that plasma FN also lacks V0-V0 dimers and consists of V0-V+ and V+-V+ combinations. Dissection of V region sequences by deletion mapping localizes the major site involved in DN dimer secretion to an 18-amino acid segment within V95. In addition, high levels of dimer secretion can be restored by insertion of V into a heterologous site 10 kD COOH terminal to its normal location. We discuss the potential role of intracellular protein-protein interactions in FN dimer formation.

scholarly journals PDZ Motifs in PTP-BL and RIL Bind to Internal Protein Segments in the LIM Domain Protein RIL

1998 ◽  
Vol 9 (3) ◽  
pp. 671-683 ◽  
Author(s):  
Edwin Cuppen ◽  
Herlinde Gerrits ◽  
Barry Pepers ◽  
Bé Wieringa ◽  
Wiljan Hendriks
Keyword(s):  
Protein Interactions ◽  
Tyrosine Phosphatase ◽  
Specific Interaction ◽  
Protein Protein Interactions ◽  
Lim Domain ◽  
Binding Interface ◽  
Mouse Tissues

The specificity of protein–protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells.

scholarly journals Chromatin Opening and Transactivator Potentiation by RAP1 in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (8) ◽  
pp. 5279-5288 ◽  
Author(s):  
Liuning Yu ◽  
Randall H. Morse
Keyword(s):  
Binding Site ◽  
Protein Interactions ◽  
Binding Sites ◽  
Accessory Protein ◽  
Dna Binding Domain ◽  
Transcriptional Activators ◽  
Protein Protein Interactions ◽  
Chromatin Opening

ABSTRACT Transcriptional activators function in vivo via binding sites that may be packaged into chromatin. Here we show that whereas the transcriptional activator GAL4 is strongly able to perturb chromatin structure via a nucleosomal binding site in yeast, GCN4 does so poorly. Correspondingly, GCN4 requires assistance from an accessory protein, RAP1, for activation of the HIS4 promoter, whereas GAL4 does not. The requirement for RAP1 for GCN4-mediated HIS4activation is dictated by the DNA-binding domain of GCN4 and not the activation domain, suggesting that RAP1 assists GCN4 in gaining access to its binding site. Consistent with this, overexpression of GCN4 partially alleviates the requirement for RAP1, whereas HIS4activation via a weak GAL4 binding site requires RAP1. RAP1 is extremely effective at interfering with positioning of a nucleosome containing its binding site, consistent with a role in opening chromatin at the HIS4 promoter. Furthermore, increasing the spacing between binding sites for RAP1 and GCN4 by 5 or 10 bp does not impair HIS4 activation, indicating that cooperative protein-protein interactions are not involved in transcriptional facilitation by RAP1. We conclude that an important role of RAP1 is to assist activator binding by opening chromatin.

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