Logesvaran Krshnan

MRC Protein Phosphorylation and Ubiquitylation Unit

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Michael L. van de Weijer

University of Oxford

Pedro Carvalho

University of Oxford

Elena Navarro‐Guerrero

University of Oxford

Matthew E. Call

University of Melbourne

Melissa Call

University of Melbourne

Jacob Robson-Tull

University of Oxford

Ulrike Grüneberg

University of Oxford

Wingyan Skyla Siu

University of Oxford

Robert Jan Lebbink

University Hospital Heidelberg

Daniel Hayward

Cancer Research UK Cambridge Center

Cooperative Institutions

MRC Protein Phosphorylation and Ubiquitylation Unit

University of Dundee

University of Oxford

Medical Research Council

Harvard University

Nanyang Technological University

Walter and Eliza Hall Institute of Medical Research

University of Melbourne

Wellcome Trust

National University of Singapore

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Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Journal of Visualized Experiments (2013)

Pooja Sharma Mariam Kaywan-Lutfi Logesvaran Krshnan Eamon F.X. Byrne Melissa Call

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Folding (DSP implementation)

Sequence (biology)

10.3791/50141

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Citations (6)

Structural Conservation and Effects of Alterations in T Cell Receptor Transmembrane Interfaces

Biophysical Journal (2018)

Soohyung Park Logesvaran Krshnan Melissa Call Matthew E. Call Wonpil Im

Structural Stability

10.1016/j.bpj.2018.01.004

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Citations (8)

Erratum: Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex (Molecular Cell (2020) 79(5) (768–781.e7), (S1097276520304755), (10.1016/j.molcel.2020.07.009))

Molecular Cell (2020)

Michael L. van de Weijer Logesvaran Krshnan S. Liberatori E. Navarro Guerrero Jacob Robson-Tull

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Regulated degradation of the inner nuclear membrane protein SUN2 maintains nuclear envelope architecture and function

eLife (2022)

Logesvaran Krshnan Wingyan Skyla Siu Michael L. van de Weijer Daniel Hayward Elena Navarro‐Guerrero

Nuclear architecture and functions depend on dynamic interactions between nuclear components (such as chromatin) and inner nuclear membrane (INM) proteins. Mutations in INM proteins interfering with these interactions result in disease. However, mechanisms controlling the levels and turnover of INM proteins remain unknown. Here, we describe a mechanism of regulated degradation of the INM SUN domain-containing protein 2 (SUN2). We show that Casein Kinase 2 and the C-terminal domain Nuclear Envelope Phosphatase 1 (CTDNEP1) have opposing effects on SUN2 levels by regulating SUN2 binding to the ubiquitin ligase Skp/Cullin1/F-Box βTrCP (SCF βTrCP ). Upon binding to phosphorylated SUN2, SCF βTrCP promotes its ubiquitination. Ubiquitinated SUN2 is membrane extracted by the AAA ATPase p97 and delivered to the proteasome for degradation. Importantly, accumulation of non-degradable SUN2 results in aberrant nuclear architecture, vulnerability to DNA damage and increased lagging chromosomes in mitosis. These findings uncover a central role of proteolysis in INM protein homeostasis.

AAA proteins

Inner membrane

Nuclear pore

Nuclear lamina

Protein Degradation

10.7554/elife.81573

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Citations (26)

A conserved αβ transmembrane interface forms the core of a compact T-cell receptor–CD3 structure within the membrane

Proceedings of the National Academy of Sciences (2016)

Logesvaran Krshnan Soohyung Park Wonpil Im Melissa Call Matthew E. Call

Significance The T-cell antigen receptor (TCR) is an eight-subunit modular biosensor that detects the presence of pathogen-derived molecular fragments in infected cells and tissues. No atomic-resolution structure has been determined for the intact receptor complex, and the mechanism by which it transmits signals across the cell membrane remains poorly defined. Using a combination of biochemical, biophysical, and computational approaches to study the assembled complex in cellular membranes, we identified an evolutionarily conserved core transmembrane (TM) structure that organizes the complex into an intimately packed eight-helix bundle within the membrane and is critical for the structural integrity of the intact receptor. Our model provides insights into possible structural pathways involved in TM TCR signaling.

10.1073/pnas.1611445113

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Citations (42)