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1.  Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6 
Bioscience Reports  2015;35(4):e00236.
CD44v6 is a co-receptor for the receptor tyrosine kinases Met and VEGFR-2 (vascular endothelial growth factor receptor 2). The binding of these RTKs (receptor tyrosine kinases) to their ligands on cells requires CD44v6. Pull-downs assays show direct binding between these entities. Binding affinities were measured by several biophysical methods.
CD44v6, a member of the CD44 family of transmembrane glycoproteins is a co-receptor for two receptor tyrosine kinases (RTKs), Met and VEGFR-2 (vascular endothelial growth factor receptor 2). CD44v6 is not only required for the activation of these RTKs but also for signalling. In order to understand the role of CD44v6 in Met and VEGFR-2 activation and signalling we tested whether CD44v6 binds to their ligands, HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor), respectively. FACS analysis and cellular ELISA showed binding of HGF and VEGF only to cells expressing CD44v6. Direct binding of CD44v6 to HGF and VEGF was demonstrated in pull-down assays and the binding affinities were determined using MicroScale Thermophoresis, fluorescence correlation spectroscopy and fluorescence anisotropy. The binding affinity of CD44v6 to HGF is in the micromolar range in contrast with the high-affinity binding measured in the case of VEGF and CD44v6, which is in the nanomolar range. These data reveal a heparan sulfate-independent direct binding of CD44v6 to the ligands of Met and VEGFR-2 and suggest different roles of CD44v6 for these RTKs.
PMCID: PMC4721543  PMID: 26181364
binding affinity; CD44s; CD44v6; HGF; Met; VEGF
2.  Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs 
The structure of VapB, a member of the Vap protein family that is involved in virulence of the bacterial pathogen R. equi, was determined by SAD phasing and reveals an eight-stranded antiparallel β-barrel similar to avidin, suggestive of a binding function. Made up of two Greek-key motifs, the topology of VapB is unusual or even unique.
Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology.
PMCID: PMC4089522  PMID: 25005079
antiparallel β-barrel; Greek-key motif; Vap protein family; single-wavelength anomalous dispersion; Rhodococcus equi
3.  Crystal structure of an engineered YopM-InlB hybrid protein 
The multi-domain protein InlB (internalin B) from Listeria monocytogenes is an agonist of the human receptor tyrosine kinase MET. Only the internalin domain directly interacts with MET. The internalin domain consists of seven central leucine-rich repeats (LRRs) flanked by an N-terminal helical cap domain and a C-terminal immunoglobulin-like structure. A potential function of the N-terminal cap in receptor binding could so far not be demonstrated by deleting the cap, since the cap is also implicated in nucleating folding of the LRR domain.
We generated an InlB variant (YopM-InlB) in which the InlB cap domain was replaced by the unrelated N-terminal capping structure of the LRR protein YopM from Yersinia enterocolitica. The crystal structure of the engineered protein shows that it folds properly. Because the first LRR is structurally closely linked to the cap domain, we exchanged LRR1 along with the cap domain. This resulted in unexpected structural changes extending to LRR2 and LRR3, which are deeply involved in MET binding. As a consequence, the binding of YopM-InlB to MET was substantially weaker than that of wild type InlB. The engineered protein was about one order of magnitude less active in colony scatter assays than wild type InlB.
We obtained a well-behaved InlB variant with an altered N-terminal capping structure through protein design. The reduced affinity for MET precludes a straightforward interpretation of the results from cell-based assays. Still, the engineered hybrid protein induced cell scatter, suggesting that the cap is required for folding and stability of InlB but is not essential for interactions that assemble the signalling-active receptor complex. The cap swap approach described here is clearly applicable to other L. monocytogenes internalins and other LRR proteins such as YopM and may yield useful structure/function correlates within this protein family.
PMCID: PMC3986869  PMID: 24669959
Capping structure; Cap domain; Chimeric protein; Hybrid protein; Internalin; Leucine-rich repeat; LRR; Protein chimera; Protein engineering; Protein stability
4.  Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells 
BMC Biophysics  2013;6:6.
The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane.
To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding.
Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.
PMCID: PMC3674922  PMID: 23731667
MET receptor; Dimerization; Single-molecule photobleaching; Fluorescence correlation spectroscopy; Fluorescence; Signal transduction
5.  Crystal structure of the Yersinia enterocolitica type III secretion chaperone SycD in complex with a peptide of the minor translocator YopD 
Type III secretion systems are used by Gram-negative bacteria as “macromolecular syringes” to inject effector proteins into eukaryotic cells. Two hydrophobic proteins called translocators form the necessary pore in the host cell membrane. Both translocators depend on binding to a single chaperone in the bacterial cytoplasm to ensure their stability and efficient transport through the secretion needle. It was suggested that the conserved chaperones bind the more divergent translocators via a hexapeptide motif that is found in both translocators and conserved between species.
We crystallized a synthetic decapeptide from the Yersinia enterocolitica minor type III secretion translocator YopD bound to its cognate chaperone SycD and determined the complex structure at 2.5 Å resolution. The structure of peptide-bound SycD is almost identical to that of apo SycD with an all helical fold consisting of three tetratricopeptide repeats (TPRs) and an additional C-terminal helix. Peptide-bound SycD formed a kinked head-to-head dimer that had previously been observed for the apo form of SycD. The homodimer interface comprises both helices of the first tetratricopeptide repeat. The YopD peptide bound in extended conformation into a mainly hydrophobic groove on the concave side of SycD. TPRs 1 and 2 of SycD form three hydrophobic pockets that accommodated the conserved hydrophobic residues at position 1, 3 and 6 of the translocator hexapeptide sequence. Two tyrosines that are highly conserved among translocator chaperones contribute to the hydrophobic patches but also form hydrogen bonds to the peptide backbone.
The interaction between SycD and YopD is very similar to the binding of the Pseudomonas minor translocator PopD to its chaperone PcrH and the Shigella major translocator IpaB to its chaperone IpgC. This confirms the prediction made by Kolbe and co-workers that a hexapeptide with hydrophobic residues at three positions is a conserved chaperone binding motif. Because the hydrophobic groove on the concave side of translocator chaperones is involved in binding of the major and the minor translocator, simultaneous binding of both translocators to a single type III secretion class II chaperone appears unlikely.
PMCID: PMC3443056  PMID: 22708907
Bacterial virulence factor; Chaperone; Complex; Crystal structure; Dimer; Peptide binding; Protein-protein interaction; Tetratricopeptide repeat; Translocator; Type III secretion
6.  Cdc42 and Phosphoinositide 3-Kinase Drive Rac-Mediated Actin Polymerization Downstream of c-Met in Distinct and Common Pathways▿ †  
Molecular and Cellular Biology  2007;27(19):6615-6628.
Activation of c-Met, the hepatocyte growth factor (HGF)/scatter factor receptor induces reorganization of the actin cytoskeleton, which drives epithelial cell scattering and motility and is exploited by pathogenic Listeria monocytogenes to invade nonepithelial cells. However, the precise contributions of distinct Rho-GTPases, the phosphatidylinositol 3-kinases, and actin assembly regulators to c-Met-mediated actin reorganization are still elusive. Here we report that HGF-induced membrane ruffling and Listeria invasion mediated by the bacterial c-Met ligand internalin B (InlB) were significantly impaired but not abrogated upon genetic removal of either Cdc42 or pharmacological inhibition of phosphoinositide 3-kinase (PI3-kinase). While loss of Cdc42 or PI3-kinase function correlated with reduced HGF- and InlB-triggered Rac activation, complete abolishment of actin reorganization and Rac activation required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N-WASP. Instead, actin polymerization was driven by Arp2/3 complex activation through the WAVE complex downstream of Rac. Together, our data establish an intricate signaling network comprising as key molecules Cdc42 and PI3-kinase, which converge on Rac-mediated actin reorganization essential for Listeria invasion and membrane ruffling downstream of c-Met.
PMCID: PMC2099217  PMID: 17682062

Results 1-6 (6)