The structural and functional characterization of the extracellular domain of vascular endothelial growth factor receptors : their role in receptor activation and use as therapeutic targets

Abstract: The vascular endothelial growth factor (VEGF) family plays key roles in the development of the blood and lymphatic vasculature. Five members, VEGF A, B, -C, -D, and PlGF can be found in the human body. They bind in an overlapping pattern to three receptor tyrosine kinases (RTKs), which constitute the type V family of RTKs: VEGF-receptor (VEGFR)-1 (also known as Flt1), VEGFR-2 (KDR/Flk1), and VEGFR-3 (Flt4). While VEGFR-1 and VEGFR-2 are mainly involved in angiogenesis, VEGFR-3 is the key player in lymphangiogenesis. VEGFRs consist of seven immunoglobulin-homology domains constituting the extracellular domain (ECD), a single transmembrane helix, and a split tyrosine kinase domain. Ligand binding to the VEGFR ectodomain initiates receptor dimerization, followed by kinase activation and autophosphorylation. Phosphorylated tyrosine residues in the intracellular domain of VEGFRs act as docking sites for a number of different signaling molecules. In addition to physiological angiogenesis, aberrant VEGFR signaling is associated with a variety of pathological conditions such as in cancer, in ischemic, and in inflammatory disorders. Several inhibitors of VEGF-signaling have been developed most of which are at different stages in clinical trials. However, anti-angiogenic treatment of cancer is often accompanied by severe side-effects and tumor patients tend to develop resistance to the treatment. Hence, structural studies of the VEGF receptor system may further elucidate the molecular mechanism underlying receptor activation and thereby help to develop new more specific drugs complementing existing therapies. During this project, I showed that binding of individual VEGFR-1 ligands resulted in conformationally similar ligand/VEGFR-1 ECD complexes. Besides showing ligand induced dimerization, the complexes reveal homotypic receptor/receptor interactions in the membrane proximal Ig homology domains. Our study is also the first addressing the thermodynamic contributions of individual Ig homology domains of VEGFR-1 to ligand binding. I showed that VEGFR-1 D4-7 positively contribute to ligand binding as shown by the higher affinities of the ligands for VEGFR-1 D1-7 compared to binding to the minimal ligand binding domain D1-3. Surprisingly, I discovered that Ig homology domain 1 blocks PlGF-1 binding to VEGFR-1 D1-3 but not to D1-7. The exact mechanism explaining this phenomenon remains unclear. In a second project, we showed that Ig-homology domains 4 and 7 are indispensable for VEGFR-2 activation. The loop connecting β-strand E and F in Ig-homology domain 7 represents the element that are required for receptor activation by mediating contacts with Ig-homology domain 7 of the second receptor chain in the dimerized complex. We generated Designed Ankyrin Repeat Proteins (DARPins) that specifically target the low affinity receptor/receptor interactions formed upon ligand binding and identified a DARPin binding to Ig-homology domain 4 that blocks VEGFR-2 activation and phosphorylation without preventing the formation of the VEGF A/VEGFR-2 complex. This inhibitor also affected downstream signaling and inhibited sprout formation of endothelial cell spheroids. This type of inhibition displays a new inhibition mechanism for VEGFR-2 that might be applied complementarily to other therapeutic approaches to improve the efficiency of anti-angiogenic therapy. ---------- Zusammenfassung: Die Familie der VEGFs spielt eine wichtige Rolle in der Entwicklung des Blut- und Lymphgefässsystems. Im menschlichen Körper sind fünf Mitglieder, VEGF A, -B, -C, -D, und PlGF anzutreffen. Sie binden in einem überlappendem Muster zu drei Rezeptor Tyrosin Kinasen, die die Typ V Familie der RTKs bilden: VEGFR-1 (auch bekannt als Flt1), VEGFR-2 (Flk1), und VEGFR-3 (Flt4). Während VEGFR-1 und VEGFR-2 hauptsächlich in der Angiogenese involviert sind, stellt VEGFR-3 eine Schlüsselfigur in der Lymphangiogenese dar. VEGFRen bestehen aus 7 Immunoglobulin-ähnlichen Dömanen in der extrazellulären Domäne, einer einzelnen membrandurchziehenden Helix, und eine geteilten intrazellulären Kinasedomäne. Ligandenbindung an die extrazelluläre Domäne initiiert Rezeptordimerisierung, gefolgt von Kinasenaktivierung und Autophosphorylierung. Phosphorylierte Tyrosinseitenketten in der intrazellulären Domäne von VEGFRen agieren als Bindestellen für eine Vielzahl von Signalmolekülen. Neben der physiologischen Angiogenese sind VEGFR-Signalwege auch in einer Vielzahl von pathologischen Konditionen involviert, z.B. Krebs, ischämischen und Entzündungskrankheiten. Eine Reihe an Inhibitoren wurde entwickelt, von denen die meisten sich in verschiedenen Stadien von klinischen Studien befinden. Allerdings wird die Anti-Angiogenese Behandlung von Krebs oft von starken Nebenwirkungen begleitet und Krebspatienten neigen dazu eine Resistenz gegen die Behandlung zu entwickeln. Daher könnten strukturelle Studien dieses Rezeptorsystems weiter dazu beitragen den molekularen Mechanismus, der der Rezeptoraktivierung unterliegt, aufzuklären, als auch helfen neue Medikamente zu entwickeln die benötigt werden um bestehende Therapien zu erweitern. Während dieses Projektes, habe ich gezeigt, dass die Bindung der einzelnen VEGFR-1 Liganden in ähnlichen Liganden/VEGFR-1 ECD Konformationen resultierte. Die Komplexe sind neben der Dimerisierung durch den Liganden durch weitere homotypische Rezeptor/Rezeptor Interaktionen in den membrannahen Ig-homologen Domänen geprägt. Ausserdem, ist dies die erste Studie, die die thermodynamische Beteiligung individueller Ig-homologie Domänen zum Prozess der Ligandenbindung behandelt. Dabei habe ich gezeigt, dass VEGFR-1 Domäne 4-7 eine positive Beteiligung am Prozess der Ligandenbindung besitzt, was durch niedrigere Affinitäten der Liganden für VEGFR-1 D1-7 im Vergleich zur minimalen Ligandenbinde Domäne gezeigt wurde. Überraschenderweise, habe ich entdeckt dass Ig-homologie Domäne 1 die PlGF-1 Bindung an VEGFR-1 D1-3 aber nicht die Bindung an D1-7 behindert. Der genaue Mechanismus, der dieses Verhalten erklären würde, ist jedoch unklar. In einem zweiten Projekt, zeigen wir dass Ig-homologie Domäne 4 und 7 unersetzlich für die VEGFR-2 Aktivierung sind. Innerhalb der Ig-homologie Domäne 7 ist es der Loop, der β-Strang E und F verbindet, der die wichtigen Elemente für die Rezeptoraktivierung beinhaltet. Dieser Loop interagiert mit demselben Loop in der Ig-homologie Domäne 7 der zweiten Rezeptorkette im dimerisierten Komplex. Daher haben wir DARPins generiert die spezifisch die niedrigaffinen Rezeptor/Rezeptor-Interaktionen anzielen, die sich durch die Ligandenbindung bilden. Wir beschreiben einen DARPin, der Ig-homologie Domäne 4 bindet und der zu einer verringerten VEGFR-2 Phosphorylierung führt ohne dabei die Ligandenbindung zu stören. Dieser Inhibitor wirkt sich auch auf Abwärtssignalwege zu PLCγ1 aus und inhibiert die Bildung von neuen Trieben von Endothelzellen. Diese Art von Inhibition stellt einen neuen Inhibitionsmechanismus für VEGFR-2 dar, der komplementär zu anderen Behandlungen benutzt werden kann um die Effizienz der Anti-Angiogenese Therapie zu verbessern

The dynamic organization of fungal acetyl-CoA carboxylase

Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control

Enzyme repurposing of a hydrolase as an emergent peroxidase upon metal binding

As an alternative to Darwinian evolution relying on catalytic promiscuity, a protein may acquire auxiliary function upon metal binding, thus providing it with a novel catalytic machinery. Here we show that addition of cupric ions to a 6-phosphogluconolactonase 6-PGLac bearing a putative metal binding site leads to the emergence of peroxidase activity (kcat7.8 × 10−2 s−1, KM 1.1 × 10−5 M). Both X-ray crystallographic and EPR data of the copper-loaded enzyme Cu·6-PGLacreveal a bis-histidine coordination site, located within a shallow binding pocket capable of accommodating the o-dianisidine substrate

Regulation of human mTOR complexes by DEPTOR

The vertebrate-specific DEP domain-containing mTOR interacting protein (DEPTOR), an oncoprotein or tumor suppressor, has important roles in metabolism, immunity, and cancer. It is the only protein that binds and regulates both complexes of mammalian target of rapamycin (mTOR), a central regulator of cell growth. Biochemical analysis and cryo-EM reconstructions of DEPTOR bound to human mTOR complex 1 (mTORC1) and mTORC2 reveal that both structured regions of DEPTOR, the PDZ domain and the DEP domain tandem (DEPt), are involved in mTOR interaction. The PDZ domain binds tightly with mildly activating effect, but then acts as an anchor for DEPt association that allosterically suppresses mTOR activation. The binding interfaces of the PDZ domain and DEPt also support further regulation by other signaling pathways. A separate, substrate-like mode of interaction for DEPTOR phosphorylation by mTOR complexes rationalizes inhibition of non-stimulated mTOR activity at higher DEPTOR concentrations. The multifaceted interplay between DEPTOR and mTOR provides a basis for understanding the divergent roles of DEPTOR in physiology and opens new routes for targeting the mTOR-DEPTOR interaction in disease

Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory “designed ankyrin repeat proteins” (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies

Real-time analysis of the binding of fluorescent VEGF₁₆₅a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis. Here we have used a novel stoichiometric protein-labeling method to generate a fluorescent variant of VEGF (VEGF₁₆₅a-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGF₁₆₅a-TMR has then been used in conjunction with full length VEGFR2, tagged with the bioluminescent protein NanoLuc, to undertake a real time quantitative evaluation of VEGFR2 binding characteristics in living cells using bioluminescence resonance energy transfer (BRET). This provided quantitative information on VEGF-VEGFR2 interactions. At longer incubation times, VEGFR2 is internalized by VEGF₁₆₅a-TMR into intracellular endosomes. This internalization can be prevented by the receptor tyrosine kinase inhibitors (RTKIs) cediranib, sorafenib, pazopanib or vandetanib. In the absence of RTKIs, the BRET signal is decreased over time as a consequence of the dissociation of agonist from the receptor in intracellular endosomes and recycling of VEGFR2 back to the plasma membrane

Collaborative Enhancement of Antibody Binding to Distinct PECAM-1 Epitopes Modulates Endothelial Targeting

Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitate targeted drug delivery to endothelial cells by “vascular immunotargeting.” To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs) to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The “collaborative enhancement” of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The “collaborative enhancement” phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents