Biology subjects

de Pereda, J. M.

Publications and source records attributed to de Pereda, J. M..

4 recordsLinked to original sources

bioRxiv2026

High-resolution cryo-EM structure of integrin αIIbβ3 bound to disease-causing maternal HPA-1a antibody that blocks integrin activation

Integrins promote immunity, embryonic development, wound healing, and hemostasis, and are activated by bent/closed to extended/open conformational changes. Integrin IIb{beta}3, being crucial for platelet activation and aggregation, is a therapeutic target for bleeding disorders and thrombosis. Human Platelet Antigen-1a (HPA-1a) on {beta}3 is recognized by pregnancy-associated maternal alloantibodies, potentially causing fetal/neonatal alloimmune thrombocytopenia (FNAIT) and even intracranial hemorrhage or perinatal death. However, severe disease determinants are largely unknown. We report the first structure of an anti-HPA-1a antibody fragment (Fab 26.4) in complex with integrin IIb{beta}3 at high resolution by cryo-electron microscopy. Fab 26.4 binding traps IIb{beta}3 in the inactive, bent/closed conformation, is incompatible with integrin extension, and inhibits IIb{beta}3-dependent fibrinogen binding and platelet aggregation. Thus, anti-HPA-1a antibodies directly impair integrin activation by preventing required conformational changes. These insights will improve FNAIT diagnostics and treatment, and spark the development of novel allosteric inhibitors against {beta}3 integrins for future therapeutic applications.

biophysics↗

bioRxiv2022

Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms

C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. Tyrosine phosphorylation of C3G and interaction with Crk adaptor proteins, whose expression is increased in multiple human cancers, participate in C3G activation. However, the molecular details of C3G activation and the interplay between C3G phosphorylation and Crk interaction are poorly understood. Here, we combine biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. CrkL interacts through its SH3N domain with the proline-rich motifs P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. Phosphorylation and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. The simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases.

molecular biology↗

bioRxiv2022

Pathogen-specific structural features of two key players in Candida albicans morphogenetic switch

Ras-like protein 1 (CaRas1) is a key regulator of the switch between the yeast and hyphal forms of Candida albicans, a feature associated with pathogenesis. CaRas1 is activated by the guanine nucleotide exchange factor (GEF) CaCdc25, triggering hyphal growth-related signaling pathways through its highly conserved GTP-binding domain (G-domain). An important function in hyphal growth has also been proposed for the long hypervariable region downstream of the G-domain of CaRas1, whose unusual content of polyQ stretches and Q/N repeats make CaRas1 unique within Ras-family proteins. Despite its biological importance, both the structure of CaRas1 and the molecular basis of its activation by CaCdc25 remain unexplored. Here, we show that CaRas1 displays an elongated shape and that its hypervariable region contains helical structural elements with intramolecular coiled-coil propensity and limited conformational flexibility. Functional assays revealed that CaRas1 activation by CaCdc25 is highly efficient, with 5-to 2000-fold higher activity levels than reported for human GEFs. In addition, the threedimensional structure of the catalytic region of CaCdc25, together with the structural characterization of CaRas1/CaCdc25 complexes, unveiled a specific region located in the -helical hairpin of CaCdc25, critical for CaRas1 activation, where negatively charged substitutions reduce its activity. The unique structural features of the low complexity region of CaRas1 and the distinctive properties of CaRas1 activation by CaCdc25, common in the homologous proteins from CTG-clade species, uncover novel strategies to target key virulence factors in human-infecting fungal pathogens.

biochemistry↗

bioRxiv2018

Structural basis of integrin α6β4 interaction with the bullous pemphigoid antigen BP230 in hemidesmosomes

Mechanical stability of epithelia requires firm attachment to the basement membrane via hemidesmosomes. Dysfunction of hemidesmosomal proteins causes severe skin blistering diseases. Two plakins, plectin and BP230 (BPAG1e), link the integrin 6{beta}4 to intermediate filaments in epidermal hemidesmosomes. Here, we show that a linear sequence within the isoform-specific N-terminal region of BP230 binds to the third and fourth FnIII domains of {beta}4. The crystal structure of the complex and mutagenesis analysis revealed that BP230 binds between the two domains of {beta}4. BP230 induces closing of the two FnIII domains that are looked in place by an inter-domain ionic clasp required for binding. Disruption of the BP230-{beta}4 interface prevents the recruitment of BP230 to hemidesmosomes in human keratinocytes, revealing a key role of the BP230-{beta}4 interaction for hemidesmosome assembly. Phosphomimetic substitutions in {beta}4 and BP230 disrupt binding. Our study provides insights into the molecular mechanisms of hemidesmosome architecture and regulation.

biophysics↗