Juha-Pekka Himanen

Adam meets Eph : An ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans

The Eph family of receptor tyrosine kinases and their ephrin ligands are mediators of cell-cell communication. Cleavage of ephrin-A2 by the ADAM10 membrane metalloprotease enables contact repulsion between Eph- and ephrin-expressing cells. How ADAM10 interacts with ephrins in a regulated manner to cleave only Eph bound ephrin molecules remains unclear. The structure of ADAM10 disintegrin and cysteine-rich domains and the functional studies presented here define an essential substrate-recognition module for functional interaction of ADAM10 with the ephrin-A5/EphA3 complex. While ADAM10 constitutively associates with EphA3, the formation of a functional EphA3/ephrin-A5 complex creates a new molecular recognition motif for the ADAM10 cysteine-rich domain that positions the proteinase domain for effective ephrin-A5 cleavage. Surprisingly, the cleavage occurs in trans, with ADAM10 and its substrate being on the membranes of opposing cells. Our data suggest a simple mechanism for regulating ADAM10-mediated ephrin proteolysis, which ensures that only Eph bound ephrins are recognized and cleaved.

Crystal structure of an Eph receptor-ephrin complex

The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are important in regulating cell–cell interactions as they initiate a unique bidirectional signal transduction cascade whereby information is communicated into both the Eph-expressing and the ephrin-expressing cells. Initially identified as regulators of axon pathfinding and neuronal cell migration, Ephs and ephrins are now known to have roles in many other cell–cell interactions, including those of vascular endothelial cells and specialized epithelia. Here we report the crystal structure of the complex formed between EphB2 and ephrin-B2, determined at 2.7 Å resolution. Each Eph receptor binds an ephrin ligand through an expansive dimerization interface dominated by the insertion of an extended ephrin loop into a channel at the surface of the receptor. Two Eph–Ephrin dimers then join to form a tetramer, in which each ligand interacts with two receptors and each receptor interacts with two ligands. The Eph and ephrin molecules are precisely positioned and orientated in these complexes, promoting higher-order clustering and the initiation of bidirectional signalling.

Eph receptors and ephrins

Eph receptors, the largest subfamily of receptor tyrosine kinases (RTKs), and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment, shape, and mobility. Eph signaling is crucial for the development of many tissues and organs including the nervous and cardiovascular systems. Both Ephs and ephrins are membrane-bound and their interactions at sites of cell-cell contact initiate unique bi-directional signaling cascades where information is transduced in both the receptor- and the ligand-expressing cells. Recent studies summarized in this review reveal how the signaling process is triggered upon ligand-receptor binding via the formation of a 2:2 circular heterotetramer. This fixes the orientation of the participating molecules and facilitates phosphorylation of their cytoplasmic domains which then interact with downstream signaling factors. The elucidation of the structural details of Eph-ephrin recognition and binding should yield insight into the future development of novel therapeutic agents targeting cardiovascular function, nerve regeneration, and cancer.

Eph signaling: a structural view

Eph receptors, the largest subfamily of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment, shape and mobility. Both Ephs and ephrins are membrane-bound and their interactions at sites of cell-cell contact initiate unique bidirectional signaling cascades, with information transduced in both the receptor-expressing and the ligand-expressing cells. Recent structural and biophysical studies summarized in this review reveal unique molecular features not previously observed in any other receptor-ligand families and explain many of the biochemical and signaling properties of Ephs and ephrins. Of particular importance is the insight into how approximation of ligand-expressing and receptor-expressing cells could lead to the formation and activation of highly ordered signaling centers at cell-cell interfaces.

Structure of the semaphorin-3A receptor binding module.

The semaphorins are a large group of extracellular proteins involved in a variety of processes during development, including neuronal migration and axon guidance. Their distinctive feature is a conserved 500 amino acid semaphorin domain, a ligand-receptor interaction module also present in plexins and scatter-factor receptors. We report the crystal structure of a secreted 65 kDa form of Semaphorin-3A (Sema3A), containing the full semaphorin domain. Unexpectedly, the semaphorin fold is a variation of the beta propeller topology. Analysis of the Sema3A structure and structure-based mutagenesis data identify the neuropilin binding site and suggest a potential plexin interaction site. Based on the structure, we present a model for the initiation of semaphorin signaling and discuss potential similarities with the signaling mechanisms of other beta propeller cell surface receptors, such as integrins and the LDL receptor.

Kinetic analysis of the binding of monomeric and dimeric ephrins to Eph receptors: Correlation to function in a growth cone collapse assay

Eph receptors and ephrins play important roles in regulating cell migration and positioning during both normal and oncogenic tissue development. Using a surface plasma resonance (SPR) biosensor, we examined the binding kinetics of representative monomeric and dimeric ephrins to their corresponding Eph receptors and correlated the apparent binding affinity with their functional activity in a neuronal growth cone collapse assay. Our results indicate that the Eph receptor binding of dimeric ephrins, formed through fusion with disulfide‐linked Fc fragments, is best described using a bivalent analyte model as a two‐step process involving an initial monovalent 2:1 binding followed by a second bivalent 2:2 binding. The bivalent binding dramatically decreases the apparent dissociation rate constants with little effect on the initial association rate constants, resulting in a 30‐ to 6000‐fold decrease in apparent equilibrium dissociation constants for the binding of dimeric ephrins to Eph receptors relative to their monomeric counterparts. Interestingly, the change was more prominent in the A‐class ephrin/Eph interactions than in the B‐class of ephrins to Eph receptors. The increase in apparent binding affinities correlated well with increased activation of Eph receptors and the resulting growth cone collapse. Our kinetic analysis and correlation of binding affinity with function helped us better understand the interactions between ephrins and Eph receptors and should be useful in the design of inhibitors that interfere with the interactions.

Mutational analysis of sickle haemoglobin (Hb) gelation

The use of recombinant Hb has provided the advantage that any amino acid substitution can be made at sites not represented by natural mutants or that cannot be modified by chemical procedures. We have recently reported the expression of human sickle Hb (HbS) in the yeast Saccharomyces cerevisiae that carries a plasmid containing the human α‐ and β‐globin cDNA sequences; N‐terminal nascent protein processing is correct and a soluble correctly folded Hb tetramer is produced. The yeast system produces a recombinant sickle Hb that is identical by about a dozen biochemical and physiological criteria with the natural sickle Hb purified from the red cells of sickle‐cell anaemia patients. Most importantly, the gelling concentration of this recombinant sickle Hb is the same as that of the HbS purified from human sickle red cells. The misfolding of Hb reported for the Escherichia coli ‐expressed protein is not apparent for Hb expressed in yeast by any of the criteria that we have used for characterization. These findings indicate that this system is well suited to the production of HbS mutants to explore those areas of the HbS tetramer whose roles in the gelation process are not yet defined and to measure quantitatively the strength of such interactions at certain inter‐tetrameric contact sites in the deoxy‐HbS aggregate. This article reviews our studies on a number of sickle Hb mutants, including polymerization‐enhancing HbS mutants and polymerization‐inhibiting HbS mutants.

STRUCTURES OF AXON GUIDANCE MOLECULES AND THEIR NEURONAL RECEPTORS

Publisher Summary This chapter discusses the structures of axon guidance molecules and their neuronal receptors. The Eph/ephrin ligand/receptor family is, by far, the best structurally characterized system. Recent structural and biochemical studies of semaphorins and of the Nogo receptor, a neuronal receptor that mediates the repulsive signals of several myelin-associated inhibitory proteins have been reviewed. The Eph receptors and the ephrins are divided into two subclasses (A and B) based on sequence conservation and their binding affinities. Ephs and ephrins also lead to transduction of a reverse signal into the ephrin-expressing cell. Eph and ephrin protein are broadly expressed during development but most abundant in the nervous system and, to a lesser extent, in vascular endothelium and specialized epithelia. The chapter discusses the structures and stoichiometry of eph/ephrin complex. The recent structural studies of Eph receptors, ephrins, and their complex suggest a likely mechanism for initiation of bi-directional signaling: Prior to cell-cell contact, Ephs and ephrins are loosely pre-clustered at the cell surface, and most likely in cholesterol-rich lipid rafts.

Structure of the ligand-binding domain of the EphB2 receptor at 2 A resolution.

Eph tyrosine kinase receptors, the largest group of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment, shape and mobility. Recently, several Eph receptors and ephrins have also been found to play important roles in the progression of cancer. Structural and biophysical studies have established detailed information on the binding and recognition of Eph receptors and ephrins. The initial high-affinity binding of Eph receptors to ephrin occurs through the penetration of an extended G-H loop of the ligand into a hydrophobic channel on the surface of the receptor. Consequently, the G-H loop-binding channel of Eph receptors is the main target in the search for Eph antagonists that could be used in the development of anticancer drugs and several peptides have been shown to specifically bind Eph receptors and compete with the cognate ephrin ligands. However, the molecular details of the conformational changes upon Eph/ephrin binding have remained speculative, since two of the loops were unstructured in the original model of the free EphB2 structure and their conformational changes upon ligand binding could consequently not be analyzed in detail. In this study, the X-ray structure of unbound EphB2 is reported at a considerably higher 2 A resolution, the conformational changes that the important receptor loops undergo upon ligand binding are described and the consequences that these findings have for the development of Eph antagonists are discussed.