Combinatorial use of disulfide bridges and native sulfur-SAD phasing for rapid structure determination of coiled-coils

Kraatz, S.H.W.; Bianchi, Sarah; Steinmetz, Michel O.

doi:10.1042/bsr20181073

Cited by 6 publications

(5 citation statements)

References 39 publications

(50 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The last chimera additionally included a F40C point mutation, located at heptad repeat position 'd' within the Gp7 cap. This mutation was made to introduce a disulphide bridge in order to facilitate experimental crystallographic phasing, since the initial location of a disulphide from anomalous diffraction data is substantially easier than of isolated sulphurs [67]. After protein expression and purification, the disulphide bridge could be confirmed by a non-reducing SDS-PAGE (data not shown).…”

Section: Crystallization and X-ray Structure Determinationmentioning

confidence: 99%

Addressing the Molecular Mechanism of Longitudinal Lamin Assembly Using Chimeric Fusions

Stalmans

Lilina

Vermeire

et al. 2020

Cells

View full text Add to dashboard Cite

The molecular architecture and assembly mechanism of intermediate filaments have been enigmatic for decades. Among those, lamin filaments are of particular interest due to their universal role in cell nucleus and numerous disease-related mutations. Filament assembly is driven by specific interactions of the elementary dimers, which consist of the central coiled-coil rod domain flanked by non-helical head and tail domains. We aimed to investigate the longitudinal ‘head-to-tail’ interaction of lamin dimers (the so-called ACN interaction), which is crucial for filament assembly. To this end, we prepared a series of recombinant fragments of human lamin A centred around the N- and C-termini of the rod. The fragments were stabilized by fusions to heterologous capping motifs which provide for a correct formation of parallel, in-register coiled-coil dimers. As a result, we established crystal structures of two N-terminal fragments one of which highlights the propensity of the coiled-coil to open up, and one C-terminal rod fragment. Additional studies highlighted the capacity of such N- and C-terminal fragments to form specific complexes in solution, which were further characterized using chemical cross-linking. These data yielded a molecular model of the ACN complex which features a 6.5 nm overlap of the rod ends.

show abstract

Section: Crystallization and X-ray Structure Determinationmentioning

confidence: 99%

Addressing the Molecular Mechanism of Longitudinal Lamin Assembly Using Chimeric Fusions

Stalmans

Lilina

Vermeire

et al. 2020

Cells

View full text Add to dashboard Cite

show abstract

“…Covalent connection of helices by a disulfide bond at its N- or C-terminus is a common approach to stabilize a coiled-coil structure. Because some of the isolated coiled-coil candidates are potentially not very stable, a second series of β3AR chimeras harboring an intramolecular disulfide bond, termed βAR3-cc_SS, was designed to increase their stability [33] (Fig. S1 and table S1).…”

Section: Resultsmentioning

confidence: 99%

A Coiled-Coil-Based Design Strategy for the Thermostabilization of G-Protein-Coupled Receptors

Amer

Frey

et al. 2022

Preprint

View full text Add to dashboard Cite

The most common methods for generating crystallizable GPCRs are scanning alanine mutagenesis and fusion to crystallization-facilitating partner proteins. The major goal of our work was to create a new GPCR tool that would provide receptor stability and additional soluble surface for crystallization. Towards this aim, we selected the two-stranded antiparallel coiled coil as a domain fold that satisfies both criteria. A selection of antiparallel coiled coils was used for structure-guided substitution of intracellular loop of the β3 adrenergic receptor. Unexpectedly, only the two GPCR variants containing thermostable coiled coils were expressed. We showed that one GPCR chimera is stable upon purification in detergent, retains ligand-binding properties, and can be crystallized. However, the quality of the crystals was not suitable for structure determination. To supply additional surface for promoting crystal contacts, we replaced in a structure-based approach the loop of the antiparallel coiled coil by T4L. Although expression is currently not suitable for structural work, we found that the engineered GPCR is even more stable than the coiled-coil variant. Our approach should be of interest for applications that benefit from stable GPCRs.

show abstract

“…At present, the instrumentation for implementing these strategies is available at only a few synchrotron facilities. A protein engineering strategy to enhance the utility of sulfur SAD involves adding rationally designed disulfide bonds ( 58 ).…”

Section: Discussionmentioning

confidence: 99%

Structural insights into plasmalemma vesicle-associated protein (PLVAP): Implications for vascular endothelial diaphragms and fenestrae

Hsieh

Smallwood

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low–molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration—presumably a coiled coil—held together with interchain disulfide bonds. Overall, ~2/3 of the ~390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP—i.e., the long dimension of the pore—and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.

show abstract

Combinatorial use of disulfide bridges and native sulfur-SAD phasing for rapid structure determination of coiled-coils

Cited by 6 publications

References 39 publications

Addressing the Molecular Mechanism of Longitudinal Lamin Assembly Using Chimeric Fusions

Addressing the Molecular Mechanism of Longitudinal Lamin Assembly Using Chimeric Fusions

A Coiled-Coil-Based Design Strategy for the Thermostabilization of G-Protein-Coupled Receptors

Structural insights into plasmalemma vesicle-associated protein (PLVAP): Implications for vascular endothelial diaphragms and fenestrae

Contact Info

Product

Resources

About