Flygaard, Habeck and Nissen question claims on bumetanide and furosemide binding to sodium-potassium-chloride cotransporter NKCC1.

Gram-negative bacteria utilize the resistance-nodulation-cell division (RND) superfamily of efflux pumps to expel a variety of toxic compounds from the cell. The Escherichia coli CusA membrane protein, which recognizes and extrudes biocidal Cu(I) and Ag(I) ions, belongs to the heavy-metal efflux (HME) subfamily of RND efflux pumps. We here report four structures of the trimeric CusA heavy-metal efflux pump in the presence of Cu(I) using single-particle cryo-electron microscopy (cryo-EM). We discover that different CusA protomers within the trimer are able to bind Cu(I) ions simultaneously. Our structural data combined with molecular dynamics (MD) simulations allow us to propose a mechanism for ion transport where each CusA protomer functions independently within the trimer.IMPORTANCE The bacterial RND superfamily of efflux pumps mediate resistance to a variety of biocides, including Cu(I) and Ag(I) ions. Here we report four cryo-EM structures of the trimeric CusA pump in the presence of Cu(I). Combined with MD simulations, our data indicate that each CusA protomer within the trimer recognizes and extrudes Cu(I) independently.

Neisseria gonorrhoeae has become a highly antimicrobial-resistant Gram-negative pathogen. Multidrug efflux is a major mechanism that N. gonorrhoeae uses to counteract the action of multiple classes of antibiotics. It appears that gonococci bearing mosaic-like sequences within the gene mtrD , encoding the most predominant and clinically important transporter of any gonococcal multidrug efflux pump, significantly elevate drug resistance and enhance transport function. Here, we report cryo-electron microscopy (EM) structures of N. gonorrhoeae MtrD carrying a mosaic-like sequence that allow us to understand the mechanism of drug recognition. Our work will ultimately inform structure-guided drug design for inhibiting these critical multidrug efflux pumps.

HSPA9, the gene coding for the mitochondrial chaperone mortalin, is involved in various cellular roles such as mitochondrial protein import, folding, degradation, Fe-S cluster biogenesis, mitochondrial homeostasis, and regulation of the antiapoptotic protein p53. Mutations in the HSPA9 gene, particularly within the region coding for the nucleotide-binding domain (NBD), cause the autosomal disorder known as EVEN-PLUS syndrome. The resulting mutants R126W and Y128C are located on the surface of the mortalin-NBD near the binding interface with the interdomain linker (IDL). We used differential scanning fluorimetry (DSF), biolayer interferometry, X-ray crystallography, ATP hydrolysis assays, and Rosetta docking simulations to study the structural and functional consequences of the EVEN-PLUS syndrome-associated R126W and Y128C mutations within the mortalin-NBD. These results indicate that the surface mutations R126W and Y128C have far-reaching effects that disrupt ATP hydrolysis, interdomain linker binding, and thermostability and increase the propensity for aggregation. The structural differences observed provide insight into how the conformations of mortalin differ from other heat shock protein 70 (Hsp70) homologues. Combined, our biophysical and structural studies contribute to the understanding of the molecular basis for how disease-associated mortalin mutations affect mortalin functionality and the pathogenesis of EVEN-PLUS syndrome.

Our early efforts to find a covalent inhibitor of mortalin, a member of the 70 kD heat shock protein (Hsp70) family, led us to solve the structure of the mortalin nucleotide‐binding domain (NBD) in complex with N6‐propargyladenosine‐5′‐diphosphate. The acquired structure emphasizes the ability of the nucleotide‐binding pocket to accommodate modified ADP compounds. A library of ADP analogs modified at either the 2‐ or N6‐positions of adenosine was screened against the mortalin‐NBD. Competitive inhibition and binding assays of the analogs demonstrate that modifications at the 2‐ or N6‐positions have potential to bind and inhibit mortalin uniquely compared to other Hsp70 homologs, and that modifications at the 2‐position confer the greatest selectivity in binding and inhibition of the mortalin‐NBD.

The heat shock protein 70, Hsp70, chaperone family plays key roles in cellular homeostasis and stress response. Hsp70s not only regulate the folding of nascent proteins, but also prevent aggregation, promote disaggregation, and refold misfolded proteins. The mitochondrial heat shock protein Mortalin functions in a variety of processes such as apoptosis, cellular stress response, mitochondrial protein import and quality control, Fe‐S cluster biogenesis, mitochondrial homeostasis, and regulation of p53, an important tumor suppressor. Mortalin is implicated in neurodegenerative diseases, and is known to play an anti‐apoptotic role in cancer cells. The Page laboratory has elucidated crystal structures of the nucleotide binding domain of human Mortalin in the apo, ADP‐bound, and AMP‐bound states at 2.8 Å, 2.78 Å, and 1.75 Å resolutions, respectively. The crystal structure of Mortalin‐NBD with the modified ADP homolog N6P‐ADP bound has also been determined at 1.49 Å. The overall structures and active site organizations are similar, with a few key differences in side chain positions. Our data identifies specific residues in the nucleotide binding pocket, which are not conserved in other Hsp70‐family members, that lead to lower nucleotide affinity and slower turnover relative to cytosolic Hsp70. Our structural data will also contribute to the understanding of disease‐associated Mortalin mutations and to improved Mortalin‐targeted compounds with the aim of influencing the function of Mortalin. Regulation of Mortalin has been proposed as a potential avenue for the treatment of cancer and neurodegenerative diseases.

Mortalin, a member of the Hsp70-family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe-S cluster protein biogenesis, mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT-077. Like other Hsp70-family members, Mortalin consists of a nucleotide-binding domain (NBD) and a substrate-binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 Å resolution. Although the Mortalin nucleotide-binding pocket is highly conserved relative to other Hsp70 family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson's disease-associated mutation is located on the Mortalin-NBD surface and may contribute to Mortalin aggregation. We present structure-based models for how the Mortalin-NBD may interact with the nucleotide exchange factor GrpEL1, with p53, and with MKT-077. Our structure may contribute to the understanding of disease-associated Mortalin mutations and to improved Mortalin-targeting antitumor compounds.