2 The abbreviations used are: c-di-AMP, cyclic di-AMP; CBS, cystathionine ␤-synthase; DRaCALA, differential radial capillary action of ligand assay; RCK, regulating conductance of K ϩ ; IPTG, isopropyl ␤-D-thiogalactopyranoside. cro ARTICLE

The multisubunit Golgi-associated retrograde protein (GARP) complex is required for tethering and fusion of endosome-derived transport vesicles to the trans-Golgi network. Mutation of leucine-967 to glutamine in the Vps54 subunit of GARP is responsible for spinal muscular atrophy in the wobbler mouse, an animal model of amyotrophic lateral sclerosis. The crystal structure at 1.7 Å resolution of the mouse Vps54 C-terminal fragment harboring leucine-967, in conjunction with comparative sequence analysis, reveals that Vps54 has a continuous α-helical bundle organization similar to that of other multisubunit tethering complexes. The structure shows that leucine-967 is buried within the α-helical bundle through predominantly hydrophobic interactions that are critical for domain stability and folding in vitro. Mutation of this residue to glutamine does not prevent integration of Vps54 into the GARP complex but greatly reduces the half-life and levels of the protein in vivo. Severely reduced levels of mutant Vps54 and, consequently, of the whole GARP complex underlie the phenotype of the wobbler mouse.Golgi apparatus | vesicle trafficking | protein stability | X-ray crystallography

P-type ATPases ubiquitously pump cations across biological membranes to maintain vital ion gradients. Among those, the chimeric K+ uptake system KdpFABC is unique. While ATP hydrolysis is accomplished by the P-type ATPase subunit KdpB, K+ has been assumed to be transported by the channel-like subunit KdpA. A first crystal structure uncovered its overall topology, suggesting such a spatial separation of energizing and transporting units. Here, we report two cryo-EM structures of the 157 kDa, asymmetric KdpFABC complex at 3.7 Å and 4.0 Å resolution in an E1 and an E2 state, respectively. Unexpectedly, the structures suggest a translocation pathway through two half-channels along KdpA and KdpB, uniting the alternating-access mechanism of actively pumping P-type ATPases with the high affinity and selectivity of K+ channels. This way, KdpFABC would function as a true chimeric complex, synergizing the best features of otherwise separately evolved transport mechanisms.

No abstract

Asparaginyl endopeptidases (AEPs) catalyze head-to-tail backbone cyclization of naturally occurring cyclic peptides such as cyclotides, and have become an important peptide-engineering tool for macrocyclization and peptide ligation. Here, we report efficient protein ligation in trans by mimicking efficient backbone cyclization by an AEP without any excess of reactants. We demonstrate a practical application of segmental isotopic labeling for NMR studies of a single-domain globular protein without any refolding step using the recombinant AEP prepared from Escherichia coli. This simple protein ligation approach using an AEP could be applied for incorporation of various biophysical probes into proteins as well as post-translational production of full-length proteins.