Biology subjects

Batista, M. R. B.

Publications and source records attributed to Batista, M. R. B..

5 recordsLinked to original sources

bioRxiv2021

Resurrecting Golgi proteins to grasp Golgi ribbon formation and self-association under stress

The Golgi complex is a membranous organelle located in the heart of the eukaryotic secretory pathway. A subfamily of the Golgi matrix proteins, called GRASPs, are key players in the stress-induced unconventional secretion, the Golgi dynamics during mitosis/apoptosis, and Golgi ribbon formation. The Golgi ribbon is vertebrate-specific and correlates with the appearance of two GRASP paralogs (GRASP55/GRASP65) and two coiled-coil Golgins (GM130/Golgin45), which interact with each other in vivo. Although essential for the Golgi ribbon formation and the increase in Golgi structural complexity, the molecular details leading to their appearance only in this subphylum are still unknown. Moreover, despite the new functionalities supported by the GRASP paralogy, little is known about the structural and evolutionary differences between these paralogues. In this context, we used ancestor sequence reconstruction and several biophysical/biochemical approaches to assess the evolution of the GRASP structure, flexibility, and how they started anchoring their Golgin partners. Our data showed that the Golgins appeared in evolution and were anchored by the single GRASP ancestor before gorasp gene duplication and divergence in Metazoans. After the gorasp divergence, variations inside the GRASP binding pocket determined which paralogue would recruit each Golgin partner (GRASP55 with Golgin45 and GRASP65 with GM130). These interactions are responsible for the proteins specific Golgi locations and the appearance of the Golgi ribbon. We also suggest that the capacity of GRASPs to form supramolecular structures is a long-standing feature, which likely affects GRASPs participation as a trigger of the stress-induced secretory pathway.

biophysics↗

bioRxiv2021

Myristoylation and its effects on the human Golgi Reassembly and Stacking Protein 55

GRASP55 is a myristoylated protein localized in the medial/trans-Golgi faces and involved in the Golgi structure maintenance and the regulation of unconventional secretion pathways. It is believed that GRASP55 achieves its main functionalities in the Golgi organization by acting as a tethering factor and, when bound to the lipid bilayer, its orientation relative to the membrane surface is restricted to determine its proper trans-oligomerization. Despite the paramount role of myristoylation in GRASP function, the impact of such protein modification on the membrane-anchoring properties and the structural organization of GRASP remains elusive. Here, an optimized protocol for the myristoylation in E. coli of the membrane-anchoring domain of GRASP55 is presented. The biophysical properties of the myristoylated/non-myristoylated GRASP55 GRASP domain were characterized in a membrane-mimicking micellar environment. Although myristoylation did not cause any impact on the proteins secondary structure, according to our circular dichroism data, it had a significant impact on the proteins thermal stability and solubility. Electrophoresis of negatively charged liposomes incubated with the two GRASP55 constructions showed different electrophoretic mobility for the myristoylated anchored protein only, thus demonstrating that myristoylation is essential for the biological membrane anchoring. Molecular dynamics simulations were used to further explore the anchoring process in determining the restricted orientation of GRASPs in the membrane.

biophysics↗

bioRxiv2021

Structural and thermodynamic analyses of human TMED1 (p24γ1) Golgi dynamics

The transmembrane emp24 domain-containing proteins (TMED), also called p24 proteins, are members of a family of sorting receptors present in all representatives of the domain Eukarya and abundantly present in all subcompartments of the early secretory pathway, namely the endoplasmic reticulum (ER), the Golgi, and the intermediate compartment. Although essential during the bidirectional transport between the ER and the Golgi, there is still a lack of information regarding the TMEDs structure, oligomerization propensity, and biophysics of their interactions with the transport cargo. Here, we describe the first high-resolution structure of the Golgi dynamics (GOLD) domain of a TMED1 representative and its biophysical characterization in solution. The crystal structure showed a dimer formation that is also present in solution in a salt-dependent manner, suggesting that the GOLD domain can form homodimers even in the absence of the TMED1 coiled-coil region. A molecular dynamics description of the dimer stabilization, with a phylogenetic analysis of the residues important for the oligomerization and a model for the orientation towards the lipid membrane are also presented.

biophysics↗

bioRxiv2019

Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix, which includes the Golgi Reassembly and Stacking Proteins (GRASPs), which participate in cisternae stacking and lateral linkage in vertebrates. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different protein binding partners. The conserved N-terminus of the GRASP family includes two PDZ domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content, however PDZ1 alone mediates nearly all the interactions between GRASPs and their binding partners. In this work, NMR, Synchrotron-Radiation Circular Dichroism and Molecular Dynamics were used to examine the structure, flexibility and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual {beta}31{beta}4{beta}52{beta}6{beta}1{beta}2 secondary structural arrangement and NMR data indicates that the PDZ1 binding pocket is formed by a stable {beta}2-strand and a more flexible and unstable 2-helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using Molecular Dynamics simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.\n\nSignificance StatementGolgi Reassembly and Stacking Proteins (GRASPs) play pivotal roles in the maintenance of Golgi structure as well as in unconventional protein secretion. Their broad network of interactions is mainly sustained by the two-PDZ domains located in the N-terminal portion of the protein. The asymmetry of the PDZ domains in terms of number and diversity of interacting partners has been long recognized, but the molecular determinants of that asymmetry remains largely unknown. The biophysical data presented here provide a firm basis for understanding why PDZ1 behaves differently to PDZ2 in solution, despite their similar 3D structures. Furthermore, we propose that PDZ2 assist ligand binding to PDZ1, by means of conformational stabilization.

biophysics↗

bioRxiv2019

Exploring Conformational Transitions and Free Energy Profiles of Proton Coupled Oligopeptide Transporters

Proteins involved in peptide uptake and transport belong to the proton-coupled oligopeptide transporter (POT) family. Crystal structures of POT family members reveal a common fold consisting of two domains of six transmembrane helices that come together to form a \"V\" shaped transporter with a central substrate binding site. Proton coupled oligopeptide transporters operate through an alternate access mechanism, where the membrane transporter undergoes global conformational changes, alternating between inward-facing (IF), outward-facing (OF) and occluded (OC) states. Conformational transitions are promoted by proton and ligand binding, however, due to the absence of crystallographic models of the outward-open state, the role of H+ and ligands are still not fully understood. To provide a comprehensive picture of the POT conformational equilibrium, conventional and enhanced sampling molecular dynamics simulations of PepTst in the presence or absence of ligand and protonation were performed. Free energy profiles of the conformational variability of PepTst were obtained from microseconds of adaptive biasing force (ABF) simulations. Our results reveal that both, proton and ligand, significantly change the conformational free energy landscape. In the absence of ligand and protonation, only transitions involving IF and OC states are allowed. After protonation of the residue Glu300, the wider free energy well for Glu300 protonated PepTst indicates a greater conformational variability relative to the apo system, and OF conformations becames accessible. For the Glu300 Holo-PepTst, the presence of a second free energy minimum suggests that OF conformations are not only accessible, but also, stable. The differences in the free energy profiles demonstrate that transitions toward outward facing conformation occur only after protonation and, probably, this should be the first step in the mechanism of peptide transport. Our extensive ABF simulations provide a fully atomic description of all states of the transport process, offering a model for the alternating access mechanism and how protonation and ligand control the conformational changes.\n\nGraphical TOC Entry\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC=\"FIGDIR/small/651646v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (21K):\norg.highwire.dtl.DTLVardef@8c8a84org.highwire.dtl.DTLVardef@da15bborg.highwire.dtl.DTLVardef@1c8dab0org.highwire.dtl.DTLVardef@18d87e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗