Employing scan-SCAM to identify residues that compose transmembrane helix 1 (TM1) of EnvZ

AbstractThe Escherichia coli sensor kinase EnvZ modulates porin expression in response to various stimuli including intracellular osmolarity, intracellular pH and periplasmic interaction with MzrA. The expression of two major outer membrane porins, OmpF and OmpC, are regulated by EnvZ, and act as passive diffusion-limited pores allowing compounds, including certain classes of antibiotics such as β-lactams and fluoroquinolones, to enter the bacterial cell. Even though allosteric processing occurs within both the periplasmic and cytoplasmic domains of EnvZ, how the transmembrane domain bi-directionally transmits these signals remains not fully understood. Here, we employ a library of single-Cys-containing EnvZ proteins to perform scan-SCAM in order to map the precise residue composition of TM1. Our results demonstrate that residue positions 19 through 30 reside within the membrane core and compose a tightly packed portion of TM1. We also show that positions 15 through 18 and position 31 are interfacial and slightly splayed apart compared to those tightly packed within the hydrophobic core. Finally, we reveal that residue positions 33 and 34 reside in the periplasm and participate in robust protein-protein interactions, while the periplasmic positions 35 through 41 exhibit helical periodicity. We conclude by synthesizing these new insights with recent high-resolution structural information into a model of membrane-spanning allosteric coupling between the periplasmic and cytoplasmic domains of EnvZ.

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Protein Interaction Domains and Post-Translational Modifications: Structural Features and Drug Discovery Applications

Current Medicinal Chemistry ◽

10.2174/0929867326666190620101637 ◽

2020 ◽

Vol 27 (37) ◽

pp. 6306-6355 ◽

Cited By ~ 2

Author(s):

Marian Vincenzi ◽

Flavia Anna Mercurio ◽

Marilisa Leone

Keyword(s):

Drug Discovery ◽

Protein Interaction ◽

Protein Interactions ◽

Structural Information ◽

Protein Complexes ◽

Structural Features ◽

Protein Protein Interactions ◽

Modular Architecture ◽

Post Translational Modifications ◽

Interaction Domains

Background:: Many pathways regarding healthy cells and/or linked to diseases onset and progression depend on large assemblies including multi-protein complexes. Protein-protein interactions may occur through a vast array of modules known as protein interaction domains (PIDs). Objective:: This review concerns with PIDs recognizing post-translationally modified peptide sequences and intends to provide the scientific community with state of art knowledge on their 3D structures, binding topologies and potential applications in the drug discovery field. Method:: Several databases, such as the Pfam (Protein family), the SMART (Simple Modular Architecture Research Tool) and the PDB (Protein Data Bank), were searched to look for different domain families and gain structural information on protein complexes in which particular PIDs are involved. Recent literature on PIDs and related drug discovery campaigns was retrieved through Pubmed and analyzed. Results and Conclusion:: PIDs are rather versatile as concerning their binding preferences. Many of them recognize specifically only determined amino acid stretches with post-translational modifications, a few others are able to interact with several post-translationally modified sequences or with unmodified ones. Many PIDs can be linked to different diseases including cancer. The tremendous amount of available structural data led to the structure-based design of several molecules targeting protein-protein interactions mediated by PIDs, including peptides, peptidomimetics and small compounds. More studies are needed to fully role out, among different families, PIDs that can be considered reliable therapeutic targets, however, attacking PIDs rather than catalytic domains of a particular protein may represent a route to obtain selective inhibitors.

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Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

eLife ◽

10.7554/elife.12509 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 56

Author(s):

Dan Tan ◽

Qiang Li ◽

Mei-Jun Zhang ◽

Chao Liu ◽

Chengying Ma ◽

...

Keyword(s):

Protein Interactions ◽

Structural Information ◽

Affinity Purification ◽

Protein Protein Interactions ◽

C Elegans ◽

Protein Protein Interaction ◽

Lysine Residues ◽

Spacer Arm ◽

Cross Linker ◽

Protein Nucleic Acid

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction.

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Activation and assembly of the NADPH oxidase: a structural perspective

Biochemical Journal ◽

10.1042/bj20041835 ◽

2005 ◽

Vol 386 (3) ◽

pp. 401-416 ◽

Cited By ~ 369

Author(s):

Yvonne GROEMPING ◽

Katrin RITTINGER

Keyword(s):

Nadph Oxidase ◽

Protein Interactions ◽

Structural Information ◽

Three Dimensional ◽

Protein Protein Interactions ◽

Crucial Component ◽

Enzyme Subunits ◽

Membrane Components ◽

Inhibitory Interactions ◽

Encompassing Model

The NADPH oxidase of professional phagocytes is a crucial component of the innate immune response due to its fundamental role in the production of reactive oxygen species that act as powerful microbicidal agents. The activity of this multi-protein enzyme is dependent on the regulated assembly of the six enzyme subunits at the membrane where oxygen is reduced to superoxide anions. In the resting state, four of the enzyme subunits are maintained in the cytosol, either through auto-inhibitory interactions or through complex formation with accessory proteins that are not part of the active enzyme complex. Multiple inputs are required to disrupt these inhibitory interactions and allow translocation to the membrane and association with the integral membrane components. Protein interaction modules are key regulators of NADPH oxidase assembly, and the protein–protein interactions mediated via these domains have been the target of numerous studies. Many models have been put forward to describe the intricate network of reversible protein interactions that regulate the activity of this enzyme, but an all-encompassing model has so far been elusive. An important step towards an understanding of the molecular basis of NADPH oxidase assembly and activity has been the recent solution of the three-dimensional structures of some of the oxidase components. We will discuss these structures in the present review and attempt to reconcile some of the conflicting models on the basis of the structural information available.

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Integrating Structural Information to Study the Dynamics of Protein-Protein Interactions in Cells

Structure ◽

10.1016/j.str.2018.07.010 ◽

2018 ◽

Vol 26 (10) ◽

pp. 1414-1424.e3 ◽

Cited By ~ 10

Author(s):

Bo Wang ◽

Zhong-Ru Xie ◽

Jiawen Chen ◽

Yinghao Wu

Keyword(s):

Protein Interactions ◽

Structural Information ◽

Protein Protein Interactions ◽

In Cells

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Protein Dynamics in F-like Bacterial Conjugation

Biomedicines ◽

10.3390/biomedicines8090362 ◽

2020 ◽

Vol 8 (9) ◽

pp. 362

Author(s):

Nicholas Bragagnolo ◽

Christina Rodriguez ◽

Naveed Samari-Kermani ◽

Alice Fours ◽

Mahboubeh Korouzhdehi ◽

...

Keyword(s):

Antibiotic Resistance ◽

Protein Interactions ◽

Drug Targets ◽

Dynamic Models ◽

Structural Information ◽

Imaging Techniques ◽

Antibiotic Resistance Genes ◽

Protein Protein Interactions ◽

Secretion Systems ◽

Type Iv

Efficient in silico development of novel antibiotics requires high-resolution, dynamic models of drug targets. As conjugation is considered the prominent contributor to the spread of antibiotic resistance genes, targeted drug design to disrupt vital components of conjugative systems has been proposed to lessen the proliferation of bacterial antibiotic resistance. Advancements in structural imaging techniques of large macromolecular complexes has accelerated the discovery of novel protein-protein interactions in bacterial type IV secretion systems (T4SS). The known structural information regarding the F-like T4SS components and complexes has been summarized in the following review, revealing a complex network of protein-protein interactions involving domains with varying degrees of disorder. Structural predictions were performed to provide insight on the dynamicity of proteins within the F plasmid conjugative system that lack structural information.

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NMR for the design of functional mimetics of protein-protein interactions: one key is in the building of bridges

Biochemistry and Cell Biology ◽

10.1139/o98-046 ◽

1998 ◽

Vol 76 (2-3) ◽

pp. 177-188 ◽

Cited By ~ 18

Author(s):

Jianxing Song ◽

Feng Ni

Keyword(s):

Protein Interactions ◽

Binding Sites ◽

Experimental Approach ◽

Structural Information ◽

Peptide Ligands ◽

Protein Protein Interactions ◽

Binding Residues ◽

Related Proteins ◽

Binding Inhibitors

Using the design of bivalent and bridge-binding inhibitors of thrombin as an example, we review an NMR-based experimental approach for the design of functional mimetics of protein-protein interactions. The strategy includes: (i) identification of binding residues in peptide ligands by differential resonance perturbation, (ii) determination of protein-bound structures of peptide ligands by use of transferred NOEs, (iii) minimization of larger protein and peptide ligands on the basis of NMR structural information, and (iv) linkage of two weakly binding mimetics to produce an inhibitor with enhanced affinity and specificity. This approach can be especially effective for the design of potent and selective functional mimetics of protein-protein interactions because it is less likely that the surfaces of two related proteins or enzymes share two identical binding sites or regions.Key words: NMR, protein-protein interactions, functional mimetics, bridge-binding inhibitors, thrombin.

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Protein–protein interactions between the bilirubin-conjugating UDPglucuronosyltransferase UGT1A1 and its shorter isoform 2 regulatory partner derived from alternative splicing

Biochemical Journal ◽

10.1042/bj20121594 ◽

2013 ◽

Vol 450 (1) ◽

pp. 107-114 ◽

Cited By ~ 5

Author(s):

Mélanie Rouleau ◽

Pierre Collin ◽

Judith Bellemare ◽

Mario Harvey ◽

Chantal Guillemette

Keyword(s):

Complex Formation ◽

Protein Interactions ◽

Disulfide Bonds ◽

Transmembrane Domain ◽

Cysteine Residues ◽

Uridine Diphosphate ◽

Protein Protein Interactions ◽

Reducing Conditions ◽

Alternatively Spliced ◽

Intermolecular Disulfide Bonds

The oligomerization of UGTs [UDP (uridine diphosphate)-glucuronosyltransferases] modulates their enzyme activities. Recent findings also indicate that glucuronidation is negatively regulated by the formation of inactive oligomeric complexes between UGT1A enzymes [i1 (isoform 1)] and an enzymatically inactive alternatively spliced i2 (isoform 2). In the present paper, we assessed whether deletion of the UGT-interacting domains previously reported to be critical for enzyme function might be involved in i1–i2 interactions. The bilirubin-conjugating UGT1A1 was used as a prototype. We also explored whether intermolecular disulfide bonds are involved in i1–i2 interactions and the potential role of selected cysteine residues. Co-immunoprecipitation assays showed that UGT1A1 lacking the SP (signal peptide) alone or also lacking the transmembrane domain (absent from i2) did not self-interact, but still interacted with i2. The deletion of other N- or C-terminal domains did not compromise i1–i2 complex formation. Under non-reducing conditions, we also observed formation of HMWCs (high-molecular-mass complexes) for cells overexpressing i1 and i2. The presence of UGTs in these complexes was confirmed by MS. Mutation of individual cysteine residues throughout UGT1A1 did not compromise i1–i1 or i1–i2 complex formation. These findings are compatible with the hypothesis that the interaction between i1 and i2 proteins (either transient or stable) involves binding of more than one domain that probably differs from those involved in i1–i1 interactions.

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A Novel Network-Based Algorithm for Predicting Protein-Protein Interactions Using Gene Ontology

Frontiers in Microbiology ◽

10.3389/fmicb.2021.735329 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lun Hu ◽

Xiaojuan Wang ◽

Yu-An Huang ◽

Pengwei Hu ◽

Zhu-Hong You

Keyword(s):

Gene Ontology ◽

Protein Interactions ◽

Structural Information ◽

Computational Prediction ◽

Biological Information ◽

Main Role ◽

Protein Protein Interactions ◽

Chronic Infections ◽

Prediction Algorithms ◽

Ppi Networks

Proteins are one of most significant components in living organism, and their main role in cells is to undertake various physiological functions by interacting with each other. Thus, the prediction of protein-protein interactions (PPIs) is crucial for understanding the molecular basis of biological processes, such as chronic infections. Given the fact that laboratory-based experiments are normally time-consuming and labor-intensive, computational prediction algorithms have become popular at present. However, few of them could simultaneously consider both the structural information of PPI networks and the biological information of proteins for an improved accuracy. To do so, we assume that the prior information of functional modules is known in advance and then simulate the generative process of a PPI network associated with the biological information of proteins, i.e., Gene Ontology, by using an established Bayesian model. In order to indicate to what extent two proteins are likely to interact with each other, we propose a novel scoring function by combining the membership distributions of proteins with network paths. Experimental results show that our algorithm has a promising performance in terms of several independent metrics when compared with state-of-the-art prediction algorithms, and also reveal that the consideration of modularity in PPI networks provides us an alternative, yet much more flexible, way to accurately predict PPIs.

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Applications of In-Cell NMR in Structural Biology and Drug Discovery

International Journal of Molecular Sciences ◽

10.3390/ijms20010139 ◽

2019 ◽

Vol 20 (1) ◽

pp. 139 ◽

Cited By ~ 9

Author(s):

CongBao Kang

Keyword(s):

Drug Discovery ◽

Conformational Changes ◽

Protein Interactions ◽

Mammalian Cells ◽

Structural Information ◽

Living Cells ◽

Bacterial Cells ◽

Protein Protein Interactions ◽

Nmr Studies ◽

Post Translational Modifications

In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein–protein interactions in living cells. Previous in-cell NMR studies have focused on proteins that were overexpressed in bacterial cells and isotopically labeled proteins injected into oocytes of Xenopus laevis or delivered into human cells. Applications of in-cell NMR in probing protein modifications, conformational changes and ligand bindings have been carried out in mammalian cells by monitoring isotopically labeled proteins overexpressed in living cells. The available protocols and successful examples encourage wide applications of this technique in different fields such as drug discovery. Despite the challenges in this method, progress has been made in recent years. In this review, applications of in-cell NMR are summarized. The successful applications of this method in mammalian and bacterial cells make it feasible to play important roles in drug discovery, especially in the step of target engagement.

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High-resolution structures of a heterochiral coiled coil

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507918112 ◽

2015 ◽

Vol 112 (43) ◽

pp. 13144-13149 ◽

Cited By ~ 18

Author(s):

David E. Mortenson ◽

Jay D. Steinkruger ◽

Dale F. Kreitler ◽

Dominic V. Perroni ◽

Gregory P. Sorenson ◽

...

Keyword(s):

Protein Interactions ◽

Structural Information ◽

Coiled Coil ◽

Coiled Coils ◽

Side Chain ◽

Amino Acid Residues ◽

Protein Protein Interactions ◽

Polypeptide Chains ◽

Packing Interactions

Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from d amino acids (d peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious protein–protein interactions. Coiled–coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled–coil interactions were predicted over 50 years ago by Crick, and limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report two independent crystal structures that elucidate coiled-coil packing between l- and d-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. However, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern.

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