Gabriela Guédez

The cell adhesion molecule L1 (L1CAM, L1 in short) plays crucial roles during neural development, regeneration after injury, synapse formation, synaptic plasticity and tumor cell migration. L1 belongs to the immunoglobulin superfamily and comprises in its extracellular part six immunoglobulin (Ig)‐like domains and five fibronectin type III homologous repeats (FNs). The second Ig‐like domain has been validated for self‐ (so‐called homophilic) binding between cells. Antibodies against this domain inhibit neuronal migration in vitro and in vivo. The fibronectin type III homologous repeats FN2 and FN3 bind small molecule agonistic L1 mimetics and contribute to signal transduction. FN3 has a stretch of 25 amino acids that can be triggered with a monoclonal antibody, or the L1 mimetics, to enhance neurite outgrowth and neuronal cell migration in vitro and in vivo. To correlate the structural features of these FNs with function, we determined a high‐resolution crystal structure of a FN2FN3 fragment, which is functionally active in cerebellar granule cells and binds several mimetics. The structure illustrates that both domains are connected by a short linker sequence allowing a flexible and largely independent organization of both domains. This becomes further evident by comparing the X‐ray crystal structure with models derived from Small‐Angle X‐ray Scattering (SAXS) data for FN2FN3 in solution. Based on the X‐ray crystal structure, we identified five glycosylation sites which we believe are crucial for folding and stability of these domains. Our study signifies an advance in the understanding of structure–functional relationships of L1.

Spermidine is a ubiquitous polyamine synthesized by spermidine synthase (SPDS) from the substrates, putrescine and decarboxylated S-adenosylmethionine (dcAdoMet). SPDS is generally active as homodimer, but higher oligomerization states have been reported in SPDS from thermophiles, which are less specific to putrescine as the aminoacceptor substrate. Several crystal structures of SPDS have been solved with and without bound substrates and/or products as well as inhibitors. Here, we determined the crystal structure of SPDS from the cyanobacterium Synechococcus (SySPDS) that is a homodimer, which we also observed in solution. Unlike crystal structures reported for bacterial and eukaryotic SPDS with bound ligands, SySPDS structure has not only bound putrescine substrate taken from the expression host, but also spermidine product most probably as a result of an enzymatic reaction. Hence, to the best of our knowledge, this is the first structure reported with both amino ligands in the same structure. Interestingly, the gate-keeping loop is disordered in the putrescine-bound monomer while it is stabilized in the spermidine-bound monomer of the SySPDS dimer. This confirms the gate-keeping loop as the key structural element that prepares the active site upon binding of dcAdoMet for the catalytic reaction of the amine donor and putrescine.

Human primary amine oxidase (hAOC3), also known as vascular adhesion protein 1, mediates leukocyte rolling and trafficking to sites of inflammation by a multistep adhesion cascade. hAOC3 is absent on the endothelium of normal tissues and is kept upregulated during inflammatory conditions, which is an applicable advantage for imaging inflammatory diseases. Sialic acid binding immunoglobulin like-lectin 9 (Siglec-9) is a leukocyte ligand for hAOC3. The peptide (CARLSLSWRGLTLCPSK) based on the region of Siglec-9 that interacts with hAOC3, can be used as a specific tracer for hAOC3-targeted imaging of inflammation using Positron Emission Tomography (PET). In the present study, we show that the Siglec-9 peptide binds to hAOC3 and triggers its amine oxidase activity towards benzylamine. Furthermore, the hAOC3 inhibitors semicarbazide and imidazole reduce the binding of wild type and Arg/Ala mutated Siglec-9 peptides to hAOC3. Molecular docking of the Siglec-9 peptide is in accordance with the experimental results and predicts that the R3 residue in the peptide interacts in the catalytic site of hAOC3 when the topaquinone cofactor is in the non-catalytic on-copper conformation. The predicted binding mode of Siglec-9 peptide to hAOC3 is supported by the PET studies using rodent, rabbit and pig AOC3 proteins.

Crystallographic snapshots illustrate the catalytic cycle and illuminate the mechanism by which the enzyme Pdx1 shuttles intermediates between lysine residues in its two active sites during the biosynthesis of pyridoxal 5′-phosphate. Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5′-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I320 intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand β6 of the Pdx1 (βα)8-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 Å between substrate- and product-binding sites.

Sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) on leukocyte surface is a counter-receptor for endothelial cell surface adhesin, human primary amine oxidase (hAOC3), a target protein for anti-inflammatory agents. This interaction can be used to detect inflammation and cancer in vivo, since the labeled peptides derived from the second C2 domain (C2₂) of Siglec-9 specifically bind to the inflammation-inducible hAOC3. As limited knowledge on the interaction between Siglec-9 and hAOC3 has hampered both hAOC3-targeted drug design and in vivo imaging applications, we have now produced and purified the extracellular region of Siglec-9 (Siglec-9-EC) consisting of the V, C2₁ and C2₂ domains, modeled its 3D structure and characterized the hAOC3–Siglec-9 interactions using biophysical methods and activity/inhibition assays. Our results assign individual, previously unknown roles for the V and C2₂ domains. The V domain is responsible for the unusually tight Siglec-9–hAOC3 interactions whereas the intact C2₂ domain of Siglec-9 is required for modulating the enzymatic activity of hAOC3, crucial for the hAOC3-mediated leukocyte trafficking. By characterizing the Siglec-9-EC mutants, we could conclude that R120 in the V domain likely interacts with the terminal sialic acids of hAOC3 attached glycans whereas residues R284 and R290 in C2₂ are involved in the interactions with the active site channel of hAOC3. Furthermore, the C2₂ domain binding enhances the enzymatic activity of hAOC3 although the sialic acid-binding capacity of the V domain of Siglec-9 is abolished by the R120S mutation. To conclude, our results prove that the V and C2₂ domains of Siglec-9-EC interact with hAOC3 in a multifaceted and unique way, forming both glycan-mediated and direct protein-protein interactions, respectively. The reported results on the mechanism of the Siglec-9–hAOC3 interaction are valuable for the development of hAOC3-targeted therapeutics and diagnostic tools.

MINT‐7994425, MINT‐7994413, MINT‐7994435: PfPdx1 (uniprotkb:C6KT50) and PfPdx1 (uniprotkb:C6KT50) bind (MI:0407) by cosedimentation in solution (MI:0028).