LINC complexes form by binding of three KASH peptides to domain interfaces of trimeric SUN proteins.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration, and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansive grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.

Structures of TorsinA and its disease-mutant complexed with an activator reveal the molecular basis for primary dystonia.

The most common cause of early onset primary dystonia, a neuromuscular disease, is a glutamate deletion (ΔE) at position 302/303 of TorsinA, a AAA+ ATPase that resides in the endoplasmic reticulum. While the function of TorsinA remains elusive, the ΔE mutation is known to diminish binding of two TorsinA ATPase activators: lamina-associated protein 1 (LAP1) and its paralog, luminal domain like LAP1 (LULL1). Using a nanobody as a crystallization chaperone, we obtained a 1.4 Å crystal structure of human TorsinA in complex with LULL1. This nanobody likewise stabilized the weakened TorsinAΔE-LULL1 interaction, which enabled us to solve its structure at 1.4 Å also. A comparison of these structures shows, in atomic detail, the subtle differences in activator interactions that separate the healthy from the diseased state. This information may provide a structural platform for drug development, as a small molecule that rescues TorsinAΔE could serve as a cure for primary dystonia.

How lamina-associated polypeptide 1 (LAP1) activates Torsin.

Lamina-associated polypeptide 1 (LAP1) resides at the nuclear envelope and interacts with Torsins, poorly understood endoplasmic reticulum (ER)-localized AAA+ ATPases, through a conserved, perinuclear domain. We determined the crystal structure of the perinuclear domain of human LAP1. LAP1 possesses an atypical AAA+ fold. While LAP1 lacks canonical nucleotide binding motifs, its strictly conserved arginine 563 is positioned exactly where the arginine finger of canonical AAA+ ATPases is found. Based on modeling and electron microscopic analysis, we propose that LAP1 targets Torsin to the nuclear envelope by forming an alternating, heterohexameric (LAP1-Torsin)3 ring, in which LAP1 acts as the Torsin activator. The experimental data show that mutation of arginine 563 in LAP1 reduces its ability to stimulate TorsinA ATPase hydrolysis. This knowledge may help scientists understand the etiology of DYT1 primary dystonia, a movement disorder caused by a single glutamate deletion in TorsinA.

Structural insights into LINC complexes.

Communication between nucleus and cytoplasm extends past molecular exchange and critically includes mechanical wiring. Cytoskeleton and nucleoskeleton are connected via molecular tethers that span the nuclear envelope. Sad1, UNC84 (SUN)-domain proteins spanning the inner nuclear membrane and Klarsicht, ANC-1 and SYNE/Nesprin-1 and -2 Homology (KASH)-peptide bearing proteins residing in the outer nuclear membrane directly bind and constitute the core of the LInkers of Nucleoskeleton and Cytoskeleton (LINC) complex. These connections appear critical for a growing number of biological processes and aberrations are implicated in a host of diverse diseases, including muscular dystrophies, cardiomyopathies, and premature aging. We discuss recent developments in this vibrant research area, particularly in context of first structural insights into LINC complexes reported in the past year.

Formation of spherical protein nanoparticles without impacting protein integrity.

Protein formulation at the nanoscale is challenging because of protein susceptibility to chemical and physical degradation during processing. Herein, we present a straightforward method to prepare spherical protein nanoparticles by co-lyophilizing five structurally different enzymes (horseradish peroxidase, carbonic anhydrase, lysozyme, subtilisin Carlsberg and α-chymotrypsin) with methyl-ß-cyclodextrin followed by suspension of the powders in ethyl acetate. The size distribution was narrow and varied from 88 ± 14 to 148 ± 16 nm as determined by dynamic light scattering. Scanning and transmission electron micrographs confirmed the size and spherical morphology of the protein nanoparticles. Residual activities for all enzymes tested were 100% upon dissolving the nanoparticles in buffer and no insoluble aggregates were formed.