Hybrid Molecules of Glutathione Reductase: Tools for Investigating Protein Interactions at the Dimer Interface

Abstract We have determined the structure of a mutant (Q237W) of HhaI DNA methyltransferase, complexed with the methyl-donor product AdoHcy. The Q237W mutant proteins were crystallized in the monoclinic space group C2 with two molecules in the crystallographic asymmetric unit. Protein-protein interface calculations in the crystal lattices suggest that the dimer interface has the specific characteristics for homodimer protein-protein interactions, while the two active sites are spatially independent on the outer surface of the dimer. The solution behavior suggests the formation of HhaI dimers as well. The same HhaI dimer interface is also observed in the previously characterized binary (M.HhaIAdoMet) and ternary (M.HhaIDNAAdoHcy) complex structures, crystallized in different space groups. The dimer is characterized either by a noncrystallographic two-fold symmetry or a crystallographic symmetry. The dimer interface involves three segments: the aminoterminal residues 28, the carboxyterminal residues 313327, and the linker (amino acids 179184) between the two functional domains the catalytic methylation domain and the DNA target recognition domain. Both the amino- and carboxy-terminal segments are part of the methylation domain. We also examined proteinprotein interactions of other structurally characterized DNA MTases, which are often found as a 2-fold related dimer with the largest dimer interface area for the group-β MTases. A possible evolutionary link between the Type I and Type II restriction-modification systems is discussed.

Download Full-text

Small Molecule–Peptide Conjugates as Dimerization Inhibitors of Leishmania infantum Trypanothione Disulfide Reductase

Pharmaceuticals ◽

10.3390/ph14070689 ◽

2021 ◽

Vol 14 (7) ◽

pp. 689

Author(s):

Alejandro Revuelto ◽

Isabel López-Martín ◽

Héctor de Lucio ◽

Juan Carlos García-Soriano ◽

Nicola Zanda ◽

...

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Leishmania Infantum ◽

Drug Target ◽

Protein Protein Interactions ◽

Peptide Conjugates ◽

Dimer Interface ◽

Disulfide Reductase ◽

Trypanosomatid Parasites ◽

Human Infections

Trypanothione disulfide reductase (TryR) is an essential homodimeric enzyme of trypanosomatid parasites that has been validated as a drug target to fight human infections. Using peptides and peptidomimetics, we previously obtained proof of concept that disrupting protein–protein interactions at the dimer interface of Leishmania infantum TryR (LiTryR) offered an innovative and so far unexploited opportunity for the development of novel antileishmanial agents. Now, we show that linking our previous peptide prototype TRL38 to selected hydrophobic moieties provides a novel series of small-molecule–peptide conjugates that behave as good inhibitors of both LiTryR activity and dimerization.

Download Full-text

Altering kinetic mechanism and enzyme stability by mutagenesis of the dimer interface of glutathione reductase

Biochemical Journal ◽

10.1042/bj3120527 ◽

1995 ◽

Vol 312 (2) ◽

pp. 527-533 ◽

Cited By ~ 20

Author(s):

A Bashir ◽

R N Perham ◽

N S Scrutton ◽

A Berry

Keyword(s):

Steady State ◽

Glutathione Reductase ◽

Enzyme Stability ◽

Alpha Helix ◽

Kinetic Mechanism ◽

Wild Type ◽

Dimer Interface ◽

Kinetic Behaviour ◽

Steady State Kinetic ◽

State Kinetic

In wild-type glutathione reductase from Escherichia coli residues Val421 and Ala422 are located in an alpha-helix in a densely packed and hydrophobic region of the dimer interface, with their side chains packed against those of residues Ala422′ and Val421′ in the second subunit. A series of mutant glutathione reductases was constructed in which the identities of the residues at positions 421 and 422 were changed. Mutations were designed so as to present like charges (mutants Val421-->Glu:Ala422-->Glu and Val421-->Lys:Ala422-->Lys) or opposite charges (mutant Val421-->Lys:Ala422-->Glu) across the dimer interface to assess the role of electrostatic interactions in dimer stability. A fourth mutant (Val421-->His:Ala422-->His) was also constructed to investigate the effects of introducing a potentially protonatable bulky side chain into a crowded region of the dimer interface. In all cases, an active dimeric enzyme was found to be assembled but each mutant protein was thermally destabilized. A detailed steady-state kinetic analysis indicated that each mutant enzyme no longer displayed the Ping Pong kinetic behaviour associated with the wild-type enzyme but exhibited what was best described as a random bireactant ternary complex mechanism. This leads, depending on the chosen substrate concentration, to apparent sigmoidal, hyperbolic or complex kinetic behaviour. These experiments, together with others reported previously, indicate that simple mutagenic changes in regions distant from the active site can lead to dramatic switches in steady-state kinetic mechanism.

Download Full-text

Human Immunodeficiency Virus Type 1 Gag Polyprotein Multimerization Requires the Nucleocapsid Domain and RNA and Is Promoted by the Capsid-Dimer Interface and the Basic Region of Matrix Protein

Journal of Virology ◽

10.1128/jvi.73.10.8527-8540.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8527-8540 ◽

Cited By ~ 142

Author(s):

Mark T. Burniston ◽

Andrea Cimarelli ◽

John Colgan ◽

Sean P. Curtis ◽

Jeremy Luban

Keyword(s):

Protein Interactions ◽

Rna Binding ◽

Protein Protein Interactions ◽

Virion Assembly ◽

Dimer Interface ◽

Gag Polyprotein ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein directs the formation of virions from productively infected cells. Manygag mutations disrupt virion assembly, but little is known about the biochemical effects of many of these mutations. Protein-protein interactions among Gag monomers are believed to be necessary for virion assembly, and data suggest that RNA may modify protein-protein interactions or even serve as a bridge linking Gag polyprotein monomers. To evaluate the primary sequence requirements for HIV-1 Gag homomeric interactions, a panel of HIV-1 Gag deletion mutants was expressed in bacteria and evaluated for the ability to associate with full-length Gag in vitro. The nucleocapsid protein, the major RNA-binding domain of Gag, exhibited activity comparable to that of the complete polyprotein. In the absence of the nucleocapsid protein, relatively weak activity was observed that was dependent upon both the capsid-dimer interface and basic residues within the matrix domain. The relevance of the in vitro findings was confirmed with an assay in which nonmyristylated mutant Gags were assessed for the ability to be incorporated into virions produced by wild-type Gag expressed intrans. Evidence of the importance of RNA for Gag-Gag interaction was provided by the demonstration that RNase impairs the Gag-Gag interaction and that HIV-1 Gag interacts efficiently with Gags encoded by distantly related retroviruses and with structurally unrelated RNA-binding proteins. These results are consistent with models in which Gag multimerization involves indirect contacts via an RNA bridge as well as direct protein-protein interactions.

Download Full-text

Structural basis of death domain signaling in the p75 neurotrophin receptor

eLife ◽

10.7554/elife.11692 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 30

Author(s):

Zhi Lin ◽

Jason Y Tann ◽

Eddy TH Goh ◽

Claire Kelly ◽

Kim Buay Lim ◽

...

Keyword(s):

Protein Interactions ◽

Binding Sites ◽

Receptor Activation ◽

Neurotrophin Receptor ◽

Structural Basis ◽

P75 Neurotrophin Receptor ◽

Death Domain ◽

Protein Protein Interactions ◽

Dimer Interface ◽

Downstream Signaling

Death domains (DDs) mediate assembly of oligomeric complexes for activation of downstream signaling pathways through incompletely understood mechanisms. Here we report structures of complexes formed by the DD of p75 neurotrophin receptor (p75NTR) with RhoGDI, for activation of the RhoA pathway, with caspase recruitment domain (CARD) of RIP2 kinase, for activation of the NF-kB pathway, and with itself, revealing how DD dimerization controls access of intracellular effectors to the receptor. RIP2 CARD and RhoGDI bind to p75NTR DD at partially overlapping epitopes with over 100-fold difference in affinity, revealing the mechanism by which RIP2 recruitment displaces RhoGDI upon ligand binding. The p75NTR DD forms non-covalent, low-affinity symmetric dimers in solution. The dimer interface overlaps with RIP2 CARD but not RhoGDI binding sites, supporting a model of receptor activation triggered by separation of DDs. These structures reveal how competitive protein-protein interactions orchestrate the hierarchical activation of downstream pathways in non-catalytic receptors.

Download Full-text

Cell Cycle–regulated Transcription in Fission Yeast: Cdc10–Res Protein Interactions during the Cell Cycle and Domains Required for Regulated Transcription

Molecular Biology of the Cell ◽

10.1091/mbc.10.11.3705 ◽

1999 ◽

Vol 10 (11) ◽

pp. 3705-3715 ◽

Cited By ~ 33

Author(s):

Simon Whitehall ◽

Peter Stacey ◽

Keren Dawson ◽

Nic Jones

Keyword(s):

Cell Cycle ◽

Protein Interactions ◽

Transcriptional Activation ◽

Regulatory Subunit ◽

Mutant Form ◽

Protein Protein Interactions ◽

Periodic Cell ◽

Binding Partners ◽

Hybrid Molecules ◽

Low Activity

In Schizosaccharomyces pombe the MBF (DSC1) complex mediates transcriptional activation at Start and is composed of a common subunit called Cdc10 in combination with two alternative DNA-binding partners, Res1 and Res2. It has been suggested that a high-activity MBF complex (at G1/S) is switched to a low-activity complex (in G2) by the incorporation of the negative regulatory subunit Res2. We have analyzed MBF protein–protein interactions and find that both Res proteins are associated with Cdc10 throughout the cell cycle, arguing against this model. Furthermore we demonstrate that Res2 is capable of interacting with a mutant form of Cdc10 that has high transcriptional activity. It has been shown previously that both Res proteins are required for periodic cell cycle–regulated transcription. Therefore a series of Res1–Res2 hybrid molecules was used to determine the domains that are specifically required to regulate periodic transcription. In Res2 the nature of the C-terminal region is critical, and in both Res1 and Res2, a domain overlapping the N-terminal ankyrin repeat and a recently identified activation domain is important for mediating cell cycle–regulated transcription.

Download Full-text

Characterization of microtubules by dark-field STEM and replication

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010008506x ◽

1991 ◽

Vol 49 ◽

pp. 152-153

Author(s):

S.B. Andrews ◽

R.D. Leapman ◽

P.E. Gallant ◽

T.S. Reese

Keyword(s):

Protein Interactions ◽

Low Dose ◽

Unit Length ◽

Dark Field ◽

Carbon Films ◽

Microtubule Associated Proteins ◽

Molecular Weights ◽

Freeze Dried ◽

Protein Motors ◽

Associated Proteins

As part of a study on protein interactions involved in microtubule (MT)-based transport, we used the VG HB501 field-emission STEM to obtain low-dose dark-field mass maps of isolated, taxol-stabilized MTs and correlated these micrographs with detailed stereo images from replicas of the same MTs. This approach promises to be useful for determining how protein motors interact with MTs. MTs prepared from bovine and squid brain tubulin were purified and free from microtubule-associated proteins (MAPs). These MTs (0.1-1 mg/ml tubulin) were adsorbed to 3-nm evaporated carbon films supported over Formvar nets on 600-m copper grids. Following adsorption, the grids were washed twice in buffer and then in either distilled water or in isotonic or hypotonic ammonium acetate, blotted, and plunge-frozen in ethane/propane cryogen (ca. -185 C). After cryotransfer into the STEM, specimens were freeze-dried and recooled to ca.-160 C for low-dose (<3000 e/nm2) dark-field mapping. The molecular weights per unit length of MT were determined relative to tobacco mosaic virus standards from elastic scattering intensities. Parallel grids were freeze-dried and rotary shadowed with Pt/C at 14°.

Download Full-text

Spot protein-protein interactions... fast

PSI Structural Genomics Knowledgebase ◽

10.1038/th_psisgkb.2010.08 ◽

2010 ◽

Author(s):

Maria Hodges

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions

Download Full-text

Role of small nuclear RNAs in eukaryotic gene expression

Essays in Biochemistry ◽

10.1042/bse0540079 ◽

2013 ◽

Vol 54 ◽

pp. 79-90 ◽

Cited By ~ 34

Author(s):

Saba Valadkhan ◽

Lalith S. Gunawardane

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Rna Stability ◽

Splice Sites ◽

Branch Site ◽

Small Nuclear Rnas ◽

Eukaryotic Gene Expression ◽

Eukaryotic Gene ◽

Rna Biogenesis

Eukaryotic cells contain small, highly abundant, nuclear-localized non-coding RNAs [snRNAs (small nuclear RNAs)] which play important roles in splicing of introns from primary genomic transcripts. Through a combination of RNA–RNA and RNA–protein interactions, two of the snRNPs, U1 and U2, recognize the splice sites and the branch site of introns. A complex remodelling of RNA–RNA and protein-based interactions follows, resulting in the assembly of catalytically competent spliceosomes, in which the snRNAs and their bound proteins play central roles. This process involves formation of extensive base-pairing interactions between U2 and U6, U6 and the 5′ splice site, and U5 and the exonic sequences immediately adjacent to the 5′ and 3′ splice sites. Thus RNA–RNA interactions involving U2, U5 and U6 help position the reacting groups of the first and second steps of splicing. In addition, U6 is also thought to participate in formation of the spliceosomal active site. Furthermore, emerging evidence suggests additional roles for snRNAs in regulation of various aspects of RNA biogenesis, from transcription to polyadenylation and RNA stability. These snRNP-mediated regulatory roles probably serve to ensure the co-ordination of the different processes involved in biogenesis of RNAs and point to the central importance of snRNAs in eukaryotic gene expression.

Download Full-text