scholarly journals A Conserved Domain in Escherichia coliLon Protease Is Involved in Substrate Discriminator Activity

1999 ◽  
Vol 181 (7) ◽  
pp. 2236-2243 ◽  
Author(s):  
Wolfgang Ebel ◽  
Monica M. Skinner ◽  
Karen P. Dierksen ◽  
Janelle M. Scott ◽  
Janine E. Trempy
Keyword(s):  
Protein Interactions ◽  
Capsular Polysaccharide ◽  
Plasmid Stability ◽  
Point Mutations ◽  
Coiled Coil ◽  
Base Change ◽  
Single Amino Acid ◽  
Protein Protein Interactions ◽  
Capsule Production ◽  
Single Amino Acid Change

ABSTRACT Lon protease of Escherichia coli regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication. Little is known about the mechanism of substrate recognition by Lon. To examine the interaction of Lon with two of its substrates, RcsA and SulA, we generated point mutations in lon which affected its substrate specificity. The most informative lon mutant overproduced capsular polysaccharide (RcsA stabilized) yet was resistant to DNA-damaging agents (SulA degraded). Immunoblots revealed that RcsA protein persisted in this mutant whereas SulA protein was rapidly degraded. The mutant contains a single-base change withinlon leading to a single amino acid change of glutamate 240 to lysine. E240 is conserved among all Lon isolates and resides in a charged domain that has a high probability of adopting a coiled-coil conformation. This conformation, implicated in mediating protein-protein interactions, appears to confer substrate discriminator activity on Lon. We propose a model suggesting that this coiled-coil domain represents the discriminator site of Lon.

scholarly journals Resilience of BST-2/Tetherin structure to single amino acid substitutions

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7043 ◽  
Author(s):  
Ian R. Roy ◽  
Camden K. Sutton ◽  
Christopher E. Berndsen
Keyword(s):  
Protein Interactions ◽  
Coiled Coil ◽  
Single Amino Acid ◽  
Missense Mutations ◽  
Protein Protein Interactions ◽  
Ensembl Database ◽  
Structural Mechanism ◽  
Important Region ◽  
Human Tetherin ◽  
Membrane Bound

Human tetherin, also known as BST-2 or CD317, is a dimeric, extracellular membrane-bound protein that consists of N and C terminal membrane anchors connected by an extracellular domain. BST-2 is involved in binding enveloped viruses, such as HIV, and inhibiting viral release in addition to a role in NF-kB signaling. Viral tethering by tetherin can be disrupted by the interaction with Vpu in HIV-1 in addition to other viral proteins. The structural mechanism of tetherin function is not clear and the effects of human tetherin mutations identified by sequencing consortiums are not known. To address this gap in the knowledge, we used data from the Ensembl database to construct and model known human missense mutations within the ectodomain to investigate how the structure of the ectodomain influences function. From the data, we identified an island of sequence stability within the ectodomain, which corresponds to a functionally and structurally important region identified in previous biochemical and biophysical studies. Most of the modeled mutations had little effect on the structure of the dimer and the coiled-coil, suggesting that the coiled-coil compensates for changes in primary structure. Thus, many of the functional defects observed in previous studies may not be due to changes in tetherin structure, but rather, due to in changes in protein-protein interactions or in aspects of tetherin not currently understood. The lack of structural effects by mutations known to decrease function further illustrates the need for more study of the structure-function connection for this system. Finally, apparent flexibility in tetherin sequence may allow for greater anti-viral activities with a larger number of viruses by reducing specific interactions with anti-tetherin proteins, while maintaining virus restriction.

scholarly journals Coiled-coil networking shapes cell molecular machinery

2012 ◽  
Vol 23 (19) ◽  
pp. 3911-3922 ◽  
Author(s):  
Yongqiang Wang ◽  
Xinlei Zhang ◽  
Hong Zhang ◽  
Yi Lu ◽  
Haolong Huang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Protein Protein Interactions ◽  
Full Spectrum ◽  
Kinetochore Assembly ◽  
Genome Wide ◽  
Molecular Machinery ◽  
Systematic Understanding

The highly abundant α-helical coiled-coil motif not only mediates crucial protein–protein interactions in the cell but is also an attractive scaffold in synthetic biology and material science and a potential target for disease intervention. Therefore a systematic understanding of the coiled-coil interactions (CCIs) at the organismal level would help unravel the full spectrum of the biological function of this interaction motif and facilitate its application in therapeutics. We report the first identified genome-wide CCI network in Saccharomyces cerevisiae, which consists of 3495 pair-wise interactions among 598 predicted coiled-coil regions. Computational analysis revealed that the CCI network is specifically and functionally organized and extensively involved in the organization of cell machinery. We further show that CCIs play a critical role in the assembly of the kinetochore, and disruption of the CCI network leads to defects in kinetochore assembly and cell division. The CCI network identified in this study is a valuable resource for systematic characterization of coiled coils in the shaping and regulation of a host of cellular machineries and provides a basis for the utilization of coiled coils as domain-based probes for network perturbation and pharmacological applications.

scholarly journals Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

2009 ◽  
Vol 284 (24) ◽  
pp. 16369-16376 ◽  
Author(s):  
Xuebo Hu ◽  
Sungkwon Kang ◽  
Xiaoyue Chen ◽  
Charles B. Shoemaker ◽  
Moonsoo M. Jin
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Coiled Coil ◽  
Affinity Maturation ◽  
Antibody Binding ◽  
Soluble Form ◽  
Protein Protein Interactions ◽  
Yeast Surface ◽  
Two Hybrid

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.

scholarly journals Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

2017 ◽  
Vol 114 (11) ◽  
pp. E2146-E2155 ◽  
Author(s):  
Chi-Yun Lin ◽  
Johan Both ◽  
Keunbong Do ◽  
Steven G. Boxer
Keyword(s):  
Quantum Yield ◽  
Protein Interactions ◽  
Fluorescent Proteins ◽  
Point Mutations ◽  
Photoactive Yellow Protein ◽  
Green Fluorescent Proteins ◽  
Protein Protein Interactions ◽  
Low Efficiency ◽  
Green Fluorescent ◽  
Rate Limiting

Split GFPs have been widely applied for monitoring protein–protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics–function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.

scholarly journals Competition NMR for Detection of Hit/Lead Inhibitors of Protein–Protein Interactions

Molecules ◽  
2020 ◽  
Vol 25 (13) ◽  
pp. 3017 ◽  
Author(s):  
Bogdan Musielak ◽  
Weronika Janczyk ◽  
Ismael Rodriguez ◽  
Jacek Plewka ◽  
Dominik Sala ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Point Mutations ◽  
Protein Protein Interactions ◽  
Competition Assay ◽  
Fragment Screening ◽  
Different Types ◽  
Lead Inhibitors

Screening for small-molecule fragments that can lead to potent inhibitors of protein–protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low μM–mM affinities. Here, we describe an NMR competition assay called w-AIDA-NMR (weak-antagonist induced dissociation assay-NMR), which is sensitive to weak μM–mM ligand–protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex’s protein that is not targeted by the inhibitor, we lower the effective affinity of the complex, allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.

scholarly journals Single Amino Acid Replacements in RocA Disrupt Protein-Protein Interactions To Alter the Molecular Pathogenesis of Group A Streptococcus

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Paul E. Bernard ◽  
Amey Duarte ◽  
Mikhail Bogdanov ◽  
James M. Musser ◽  
Randall J. Olsen
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Molecular Pathogenesis ◽  
Single Amino Acid ◽  
Accessory Protein ◽  
Protein Protein Interactions ◽  
Tissue Destruction ◽  
Intact Cells ◽  
Group A

ABSTRACT Group A Streptococcus (GAS) is a human-specific pathogen and major cause of disease worldwide. The molecular pathogenesis of GAS, like many pathogens, is dependent on the coordinated expression of genes encoding different virulence factors. The control of virulence regulator/sensor (CovRS) two-component system is a major virulence regulator of GAS that has been extensively studied. More recent investigations have also involved regulator of Cov (RocA), a regulatory accessory protein to CovRS. RocA interacts, in some manner, with CovRS; however, the precise molecular mechanism is unknown. Here, we demonstrate that RocA is a membrane protein containing seven transmembrane helices with an extracytoplasmically located N terminus and cytoplasmically located C terminus. For the first time, we demonstrate that RocA directly interacts with itself (RocA) and CovS, but not CovR, in intact cells. Single amino acid replacements along the entire length of RocA disrupt RocA-RocA and RocA-CovS interactions to significantly alter the GAS virulence phenotype as defined by secreted virulence factor activity in vitro and tissue destruction and mortality in vivo. In summary, we show that single amino acid replacements in a regulatory accessory protein can affect protein-protein interactions to significantly alter the virulence of a major human pathogen.

scholarly journals Divisome under Construction: Distinct Domains of the Small Membrane Protein FtsB Are Necessary for Interaction with Multiple Cell Division Proteins

2009 ◽  
Vol 191 (8) ◽  
pp. 2815-2825 ◽  
Author(s):  
Mark D. Gonzalez ◽  
Jon Beckwith
Keyword(s):  
Cell Division ◽  
Membrane Proteins ◽  
Protein Interactions ◽  
Cell Envelope ◽  
Coiled Coil ◽  
Main Function ◽  
Protein Protein Interactions ◽  
Molecular Scaffold ◽  
C Terminus ◽  
Under Construction

ABSTRACT Cell division in bacteria requires the coordinated action of a set of proteins, the divisome, for proper constriction of the cell envelope. Multiple protein-protein interactions are required for assembly of a stable divisome. Within the Escherichia coli divisome is a conserved subcomplex of inner membrane proteins, the FtsB/FtsL/FtsQ complex, which is necessary for linking the upstream division proteins, which are predominantly cytoplasmic, with the downstream division proteins, which are predominantly periplasmic. FtsB and FtsL are small bitopic membrane proteins with predicted coiled-coil motifs, which themselves form a stable subcomplex that can recruit downstream division proteins independently of FtsQ; however, the details of how FtsB and FtsL interact together and with other proteins remain to be characterized. Despite the small size of FtsB, we identified separate interaction domains of FtsB that are required for interaction with FtsL and FtsQ. The N-terminal half of FtsB is necessary for interaction with FtsL and sufficient, when in complex with FtsL, for recruitment of downstream division proteins, while a portion of the FtsB C terminus is necessary for interaction with FtsQ. These properties of FtsB support the proposal that its main function is as part of a molecular scaffold to allow for proper formation of the divisome.

scholarly journals Crystal Structure of SEDL and Its Implications for a Genetic Disease Spondyloepiphyseal Dysplasia Tarda

2002 ◽  
Vol 277 (51) ◽  
pp. 49863-49869 ◽  
Author(s):  
Se Bok Jang ◽  
Yeon-Gil Kim ◽  
Yong-Soon Cho ◽  
Pann-Ghill Suh ◽  
Kyung-Hwa Kim ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Genetic Disease ◽  
Point Mutations ◽  
Missense Mutations ◽  
Protein Protein Interactions ◽  
Spondyloepiphyseal Dysplasia ◽  
Multiple Protein ◽  
Protein Particle ◽  
Eukaryotic Organisms ◽  
Spondyloepiphyseal Dysplasia Tarda

SEDL is an evolutionarily highly conserved protein in eukaryotic organisms. Deletions or point mutations in theSEDLgene are responsible for the genetic disease spondyloepiphyseal dysplasia tarda (SEDT), an X-linked skeletal disorder. SEDL has been identified as a component of the transport protein particle (TRAPP), critically involved in endoplasmic reticulum-to-Golgi vesicle transport. Herein, we report the 2.4 Å resolution structure of SEDL, which reveals an unexpected similarity to the structures of the N-terminal regulatory domain of two SNAREs, Ykt6p and Sec22b, despite no sequence homology to these proteins. The similarity and the presence of unusually many solvent-exposed apolar residues of SEDL suggest that it serves regulatory and/or adaptor functions through multiple protein-protein interactions. Of the four known missense mutations responsible for SEDT, three mutations (S73L, F83S, V130D) map to the protein interior, where the mutations would disrupt the structure, and the fourth (D47Y) on a surface at which the mutation may abrogate functional interactions with a partner protein.

scholarly journals Protein-protein interactions can be predicted using coiled coil co-evolution patterns

2017 ◽  
Vol 412 ◽  
pp. 198-203 ◽  
Author(s):  
Pablo Mier ◽  
Gregorio Alanis-Lobato ◽  
Miguel A. Andrade-Navarro
Keyword(s):  
Protein Interactions ◽  
Coiled Coil ◽  
Protein Protein Interactions

scholarly journals Structure of the Notch1-negative regulatory region: implications for normal activation and pathogenic signaling in T-ALL

Blood ◽  
2009 ◽  
Vol 113 (18) ◽  
pp. 4381-4390 ◽  
Author(s):  
Wendy R. Gordon ◽  
Monideepa Roy ◽  
Didem Vardar-Ulu ◽  
Megan Garfinkel ◽  
Marc R. Mansour ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Structural Integrity ◽  
Crystal Packing ◽  
Novel Mutation ◽  
Lymphoblastic Leukemia ◽  
Regulatory Region ◽  
Point Mutations ◽  
Protein Protein Interactions ◽  
Proteolytic Resistance ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Proteolytic resistance of Notch prior to ligand binding depends on the structural integrity of a negative regulatory region (NRR) of the receptor that immediately precedes the transmembrane segment. The NRR includes the 3 Lin12/Notch repeats and the juxtamembrane heterodimerization domain, the region of Notch1 most frequently mutated in T-cell acute lymphoblastic leukemia lymphoma (T-ALL). Here, we report the x-ray structure of the Notch1 NRR in its autoinhibited conformation. A key feature of the Notch1 structure that maintains its closed conformation is a conserved hydrophobic plug that sterically occludes the metalloprotease cleavage site. Crystal packing interactions involving a highly conserved, exposed face on the third Lin12/Notch repeat suggest that this site may normally be engaged in intermolecular or intramolecular protein-protein interactions. The majority of known T-ALL–associated point mutations map to residues in the hydrophobic interior of the Notch1 NRR. A novel mutation (H1545P), which alters a residue at the crystal-packing interface, leads to ligand-independent increases in signaling in reporter gene assays despite only mild destabilization of the NRR, suggesting that it releases the autoinhibitory clamp on the heterodimerization domain imposed by the Lin12/Notch repeats. The Notch1 NRR structure should facilitate a search for antibodies or compounds that stabilize the autoinhibited conformation.

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