Crystal structure of the second extracellular domain of human tetraspanin CD9: twinning and diffuse scattering

Remarkable features are reported in the diffraction pattern produced by a crystal of the second extracellular domain of tetraspanin CD9 (deemed CD9EC2), the structure of which has been described previously [Oosterheert et al. (2020[Oosterheert, W., Xenaki, K. T., Neviani, V., Pos, W., Doulkeridou, S., Manshande, J., Pearce, N. M., Kroon-Batenburg, L. M. J., Lutz, M., van Bergen en Henegouwen, P. M. P. & Gros, P. (2020). Life Sci. Alliance, 3, e202000883.]), Life Sci. Alliance, 3, e202000883]. CD9EC2 crystallized in space group P1 and was twinned. Two types of diffuse streaks are observed. The stronger diffuse streaks are related to the twinning and occur in the direction perpendicular to the twinning interface. It is concluded that the twin domains scatter coherently as both Bragg reflections and diffuse streaks are seen. The weaker streaks along c* are unrelated to the twinning but are caused by intermittent layers of non-crystallographic symmetry related molecules. It is envisaged that the raw diffraction images could be very useful for methods developers trying to remove the diffuse scattering to extract accurate Bragg intensities or using it to model the effect of packing disorder on the molecular structure

Multifaceted Activities of Seven Nanobodies against Complement C4b

Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. Dysregulated activation of C4 and other initial components of the classical pathway may cause or aggravate pathologies, such as systemic lupus erythematosus, Alzheimer disease, and schizophrenia. Modulating the activity of C4b by small-molecule or protein-based inhibitors may represent a promising therapeutic approach for preventing excessive inflammation and damage to host cells and tissue. Here, we present seven nanobodies, derived from llama (Lama glama) immunization, that bind to human C4b (Homo sapiens) with high affinities ranging from 3.2 nM to 14 pM. The activity of the nanobodies varies from no to complete inhibition of the classical pathway. The inhibiting nanobodies affect different steps in complement activation, in line with blocking sites for proconvertase formation, C3 substrate binding to the convertase, and regulator-mediated inactivation of C4b. For four nanobodies, we determined singleparticle cryo-electron microscopy structures in complex with C4b at 3.4-4 Å resolution. The structures rationalize the observed functional effects of the nanobodies and define their mode of action during complement activation. Thus, we characterized seven anti-C4b nanobodies with diverse effects on the classical pathway of complement activation that may be explored for imaging, diagnostic, or therapeutic applications

Molecular mechanisms of tumor-cell markers: Structural insights into the STEAP and tetraspanin membrane protein families

Cancer is the collective name for more than 200 diseases which are characterized by uncontrolled growth and division of a group of cells in the human body. If these cells form a mass with the potential to invade other parts of the body, this cell mass is defined as a malignant tumor. The molecular landscape of tumor-cell membranes is formed by proteins that stimulate the growth and survival of the tumor cell. These membrane proteins are classified as tumor markers and are important targets for cancer immunotherapy. In this PhD research, we investigated two membrane protein families, the ‘six-transmembrane epithelial antigen of the prostate’ proteins (STEAPs) and tetraspanins. Both STEAPs and tetraspanins are highly upregulated on the membranes of several tumors, but at the start of our research there were limited insights into the molecular mechanisms of these proteins. Thus, the goal of our research was to increase our understanding of the molecular mechanisms of STEAPs and tetraspanins, with the ultimate aim to allow for the design of new translational research strategies to target these membrane proteins in cancer. We solved the 3D structures of STEAPs and of the tetraspanin CD9 using cryo-electron microscopy. The function of STEAPs is to reduce iron and copper. The STEAP-structures allowed us to generate a model of how STEAPs transport electrons from inside the cell to extracellular metal ions. These molecular insights into the metalloreductase activity of STEAPs may be useful in the design of new therapeutic strategies to target STEAPs in cancer. Tetraspanins are known as ‘molecular organizers’ of the cell membrane because they cluster specific partner proteins in microdomains. Our structure of tetraspanin CD9 in complex with its partner protein EWI-F provide implications for the assembly of these microdomains

Molecular mechanisms of tumor-cell markers: Structural insights into the STEAP and tetraspanin membrane protein families

Cancer is the collective name for more than 200 diseases which are characterized by uncontrolled growth and division of a group of cells in the human body. If these cells form a mass with the potential to invade other parts of the body, this cell mass is defined as a malignant tumor. The molecular landscape of tumor-cell membranes is formed by proteins that stimulate the growth and survival of the tumor cell. These membrane proteins are classified as tumor markers and are important targets for cancer immunotherapy. In this PhD research, we investigated two membrane protein families, the ‘six-transmembrane epithelial antigen of the prostate’ proteins (STEAPs) and tetraspanins. Both STEAPs and tetraspanins are highly upregulated on the membranes of several tumors, but at the start of our research there were limited insights into the molecular mechanisms of these proteins. Thus, the goal of our research was to increase our understanding of the molecular mechanisms of STEAPs and tetraspanins, with the ultimate aim to allow for the design of new translational research strategies to target these membrane proteins in cancer. We solved the 3D structures of STEAPs and of the tetraspanin CD9 using cryo-electron microscopy. The function of STEAPs is to reduce iron and copper. The STEAP-structures allowed us to generate a model of how STEAPs transport electrons from inside the cell to extracellular metal ions. These molecular insights into the metalloreductase activity of STEAPs may be useful in the design of new therapeutic strategies to target STEAPs in cancer. Tetraspanins are known as ‘molecular organizers’ of the cell membrane because they cluster specific partner proteins in microdomains. Our structure of tetraspanin CD9 in complex with its partner protein EWI-F provide implications for the assembly of these microdomains

An Elegant Four-Helical Fold in NOX and STEAP Enzymes Facilitates Electron Transport across Biomembranes—Similar Vehicle, Different Destination

The ferric reductase superfamily comprises several oxidoreductases that use an intracellular electron source to reduce an extracellular acceptor substrate. NADPH oxidases (NOXs) and six-transmembrane epithelial antigen of the prostate enzymes (STEAPs) are iconic members of the superfamily. NOXs produce extracellular reactive oxygen species that exert potent bactericidal activities and trigger redox-signaling cascades that regulate cell division and differentiation. STEAPs catalyze the reduction of extracellular iron and copper which is necessary for the bioavailability of these essential elements. Both NOXs and STEAPs are present as multiple isozymes with distinct regulatory properties and physiological roles. Despite the important roles of NOXs and STEAPs in human physiology and despite their wide involvement in diseases like cancer, their mode of action at the molecular level remained incompletely understood for a long time, in part due to the absence of high-resolution models of the complete enzymes. Our two laboratories have elucidated the three-dimensional structures of NOXs and STEAPs, providing key insight into their mechanisms and evolution. The enzymes share a conserved transmembrane helical domain with an eye-catching hourglass shape. On the extracellular side, a heme prosthetic group is at the bottom of a pocket where the substrate (

Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments

The release of inorganic phosphate (Pi) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. However, the molecular mechanisms underlying Pi release from both the core and barbed end of actin filaments remain unclear. Here, we combine cryo-EM with molecular dynamics simulations and in vitro reconstitution to demonstrate how actin releases Pi through a ‘molecular backdoor’. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and only opens transiently through concerted backbone movements and rotameric rearrangements of residues close to the nucleotide binding pocket. This mechanism explains why Pi escapes rapidly from the filament end and yet slowly from internal actin subunits. In an actin variant associated with nemaline myopathy, the backdoor is predominantly open in filament-core subunits, resulting in greatly accelerated Pi release after polymerization and filaments with drastically shortened ADP-Pi caps. This demonstrates that the Pi release rate from F-actin is controlled by steric hindrance through the backdoor rather than by the disruption of the ionic bond between Pi and Mg2+ at the nucleotide-binding site. Our results provide the molecular basis for Pi release from actin and exemplify how a single, disease-linked point mutation distorts the nucleotide state distribution and atomic structure of the actin filament

Cryo-EM structures of human STEAP4 reveal mechanism of iron(III) reduction

Enzymes of the six-transmembrane epithelial antigen of the prostate (STEAP) family reduce Fe3+ and Cu2+ ions to facilitate metal-ion uptake by mammalian cells. STEAPs are highly upregulated in several types of cancer, making them potential therapeutic targets. However, the structural basis for STEAP-catalyzed electron transfer through an array of cofactors to metals at the membrane luminal side remains elusive. Here, we report cryo-electron microscopy structures of human STEAP4 in absence and presence of Fe3+-NTA. Domain-swapped, trimeric STEAP4 orients NADPH bound to a cytosolic domain onto axially aligned flavin-adenine dinucleotide (FAD) and a single b-type heme that cross the transmembrane-domain to enable electron transfer. Substrate binding within a positively charged ring indicates that iron gets reduced while in complex with its chelator. These molecular principles of iron reduction provide a basis for exploring STEAPs as therapeutic targets

Cryo-EM structures of human STEAP4 reveal mechanism of iron(III) reduction

Enzymes of the six-transmembrane epithelial antigen of the prostate (STEAP) family reduce Fe3+ and Cu2+ ions to facilitate metal-ion uptake by mammalian cells. STEAPs are highly upregulated in several types of cancer, making them potential therapeutic targets. However, the structural basis for STEAP-catalyzed electron transfer through an array of cofactors to metals at the membrane luminal side remains elusive. Here, we report cryo-electron microscopy structures of human STEAP4 in absence and presence of Fe3+-NTA. Domain-swapped, trimeric STEAP4 orients NADPH bound to a cytosolic domain onto axially aligned flavin-adenine dinucleotide (FAD) and a single b-type heme that cross the transmembrane-domain to enable electron transfer. Substrate binding within a positively charged ring indicates that iron gets reduced while in complex with its chelator. These molecular principles of iron reduction provide a basis for exploring STEAPs as therapeutic targets

Crystal structure of the second extracellular domain of human tetraspanin CD9: twinning and diffuse scattering

Remarkable features are reported in the diffraction pattern produced by a crystal of the second extracellular domain of tetraspanin CD9 (deemed CD9EC2), the structure of which has been described previously [Oosterheert et al. (2020[Oosterheert, W., Xenaki, K. T., Neviani, V., Pos, W., Doulkeridou, S., Manshande, J., Pearce, N. M., Kroon-Batenburg, L. M. J., Lutz, M., van Bergen en Henegouwen, P. M. P. & Gros, P. (2020). Life Sci. Alliance, 3, e202000883.]), Life Sci. Alliance, 3, e202000883]. CD9EC2 crystallized in space group P1 and was twinned. Two types of diffuse streaks are observed. The stronger diffuse streaks are related to the twinning and occur in the direction perpendicular to the twinning interface. It is concluded that the twin domains scatter coherently as both Bragg reflections and diffuse streaks are seen. The weaker streaks along c* are unrelated to the twinning but are caused by intermittent layers of non-crystallographic symmetry related molecules. It is envisaged that the raw diffraction images could be very useful for methods developers trying to remove the diffuse scattering to extract accurate Bragg intensities or using it to model the effect of packing disorder on the molecular structure