The two-domain elevator-type mechanism of zinc-transporting ZIP proteins.

Zinc is essential for all organisms and yet detrimental at elevated levels. Hence, homeostasis of this metal is tightly regulated. The Zrt/Irt-like proteins (ZIPs) represent the only zinc importers in metazoans. Mutations in human ZIPs cause serious disorders, but the mechanism by which ZIPs transfer zinc remains elusive. Hitherto, structural information is only available for a model member, BbZIP, and as a single, ion-bound conformation, precluding mechanistic insights. Here, we elucidate an inward-open metal-free BbZIP structure, differing substantially in the relative positions of the two separate domains of ZIPs. With accompanying coevolutional analyses, mutagenesis, and uptake assays, the data point to an elevator-type transport mechanism, likely shared within the ZIP family, unifying earlier functional data. Moreover, the structure reveals a previously unknown ninth transmembrane segment that is important for activity in vivo. Our findings outline the mechanistic principles governing ZIP-protein transport and enhance the molecular understanding of ZIP-related disorders.

Saccharomyces cerevisiae as a superior host for overproduction of prokaryotic integral membrane proteins.

Integral membrane proteins (IMPs) constitute ~30% of all proteins encoded by the genome of any organism and Escherichia coli remains the first-choice host for recombinant production of prokaryotic IMPs. However, the expression levels of prokaryotic IMPs delivered by this bacterium are often low and overproduced targets often accumulate in inclusion bodies. The targets are therefore often discarded to avoid an additional and inconvenient refolding step in the purification protocol. Here we compared expression of five prokaryotic (bacterial and archaeal) IMP families in E. coli and Saccharomyces cerevisiae. We demonstrate that our S. cerevisiae-based production platform is superior in expression of four investigated IMPs, overall being able to deliver high quantities of active target proteins. Surprisingly, in case of the family of zinc transporters (Zrt/Irt-like proteins, ZIPs), S. cerevisiae rescued protein expression that was undetectable in E. coli. We also demonstrate the effect of localization of the fusion tag on expression yield and sample quality in detergent micelles. Lastly, we present a road map to achieve the most efficient expression of prokaryotic IMPs in our yeast platform. Our findings demonstrate the great potential of S. cerevisiae as host for high-throughput recombinant overproduction of bacterial and archaeal IMPs for downstream biophysical characterization.

Structure-Based Design of a New Scaffold for Cell-Penetrating Peptidic Inhibitors of the Histone Demethylase PHF8.

The histone demethylase PHF8 catalyzes demethylation of mono- and di-methylated Lys9 on histone H3 (H3K9me1/2), and is a transcriptional activator involved in the development and cancer. Affinity and specificity of PHF8 towards H3K9me2 is affected by interaction with both the catalytic domain and a PHD reader domain. The latter specifically recognizes tri-methylated Ly4 on histone H3. A fragment of the histone H3 tail with tri-methylated Lys4 was used as a template for the structure-based design of a cyclic, cell-penetrating peptide that exhibits micromolar binding affinity to PHF8 in biochemical assays. The inhibitor has significantly lower affinity towards KDM2 enzymes (the phylogenetically closest subfamily), and to KDM3 and KDM6 subfamilies. Selectivity is only marginal towards an enzyme from the KDM4 family, which shares histone tail specificity with PHF8. It is a substrate of KDM5B, thus implying that the free N terminus is not part of the KDM5 enzyme substrate recognition machinery. The cyclic peptide's ability to penetrate cells is achieved by incorporation of a sequence derived from HIV Tat. The derived cyclic peptide can be used as a starting compound in the search for potent and selective PHF8 inhibitors.

Expression, purification and characterization of the human MTA2-RBBP7 complex.

The repressive Nucleosome Remodeling and histone Deacetylation (NuRD) complex remodels the chromatin structure by coupling ATP-dependent remodeling activity with histone deacetylase function and plays important roles in regulating gene transcription, DNA damage repair and chromatin assembly. The complex is composed of six subunits: Metastasis Associated proteins MTA1/2/3 initially recruit histone chaperones RBBP4/7 followed by the histone deacetylases HDAC1/2 forming a core complex. Further association of the CpG-binding protein MBD2/3, p66α/ß and the ATP-dependent helicase CDH3/4 constitutes the NuRD complex. Recent structural studies on truncated human proteins or orthologous have revealed that the stoichiometry of the MTA1-RBBP4 complex is 2:4. This study reports expression and purification of the intact human MTA2-RBBP7 complex using HEK293F cells as expression system. In analogy with findings on the Drosophila NuRD complex, we find that also the human MTA-RBBP can be isolated in vitro. Taken together with previous findings this suggests, that MTA-RBBP is a stable complex, with a central role in the initial assembly of the human NuRD complex. Refined 3D volumes of the complex generated from negative stain electron microscopy (EM) data reveals an elongated architecture that is capable of hinge like motion around the center of the particle.

Structure and binding properties of a cameloid nanobody raised against KDM5B.

The histone demethylase KDM5B is considered to be a promising target for anticancer therapy. Single-chain antibodies from llama (nanobodies) have been raised to aid in crystallization and structure determination of this enzyme. The antigen-binding properties of 15 of these nanobodies have been characterized. The crystal structure of one of these (NB17) has been determined to a resolution of 1.85 Å. NB17 crystallizes in space group P4322 with six molecules in the asymmetric unit. The six molecules in the asymmetric unit pack as an entity with approximate D3 symmetry with interactions mediated by the CDR loops, which could act as a crystallization nucleus. NB17 does not bind to monomeric KDM5B residues 1-820, but is found to bind to aggregates formed after incubation at 310 K.