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Journal of cell science 133(19), jcs239202 (1-15) - (2020). doi:10.1242/jcs.239202, Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the �� integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn$^{2+}$. Here we show that kindlin-1 can replace Mn2+ to mediate ��3 integrin clustering induced by the talin head, but not that induced by the F2���F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for ��3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of ��- and ��3-integrins, in order to activate the ��3 integrin heterodimer, further detailing the mechanism by which the talin���kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the ��3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer., Published by Company of Biologists Limited, Cambridge

Cell walls of marine macroalgae are composed of diverse polysaccharides that provide abundant carbon sources for marine heterotrophic bacteria. Among them, Zobellia galactanivorans is considered as a model for studying algae–bacteria interactions. The degradation of typical algal polysaccharides, such as agars or alginate, has been intensively studied in this model bacterium, but the catabolism of plant-like polysaccharides is essentially uncharacterized. Here, we identify a polysaccharide utilization locus in the genome of Z. galactanivorans, induced by laminarin (β-1,3-glucans), and containing a putative GH5 subfamily 4 (GH5_4) enzyme, currently annotated as a endoglucanase (ZgEngAGH5_4). A phylogenetic analysis indicates that ZgEngAGH5_4 was laterally acquired from an ancestral Actinobacteria. We performed the biochemical and structural characterization of ZgEngAGH5_4 and demonstrated that this GH5 is, in fact, an endo-β-glucanase, most active on mixed-linked glucan (MLG). Although ZgEngAGH5_4 and GH16 lichenases both hydrolyze MLG, these two types of enzymes release different series of oligosaccharides. Structural analyses of ZgEngAGH5_4 reveal that all the amino acid residues involved in the catalytic triad and in the negative glucose-binding subsites are conserved, when compared with the closest relative, the cellulase EngD from Clostridium cellulovorans, and some other GH5s. In contrast, the positive glucose-binding subsites of ZgEngAGH5_4 are different and this could explain the preference for MLG, with respect to cellulose or laminarin. Molecular dynamics computer simulations using different hexaoses reveal that the specificity for MLG occurs through the +1 and +2 subsites of the binding pocket that display the most important differences when compared with the structures of other GH5_4 enzymes.

It has been a long-standing question how the two leaflets in a lipid bilayer modulate each others' physical properties. In this paper, we discuss how this interaction may take place through interdigitation. We use atomistic molecular dynamics simulations to consider asymmetric lipid membrane models whose compositions are based on the lipidomics data determined for exosomes released by PC-3 prostate cancer cells. The simulations show interdigitation to be exceptionally strong for long-chain sphingomyelin (SM) molecules. In asymmetric membranes the amide-linked chain of SM is observed to extend deep into the opposing membrane leaflet. Interestingly, we find that the conformational order of the amide-linked SM chain increases the deeper it penetrates to the opposing leaflet. Analysis of this finding reveals that the amide-linked SM chain interacts favorably with the lipid chains in the opposite leaflet, and that cholesterol modulates the effect of SM interdigitation by influencing the conformational order of lipid hydrocarbon chains in the opposing (cytosolic) leaflet.

peer-reviewed Transformation of cellulose into monosaccharides can be achieved by hydrolysis of the cellulose chains, carried out by a special group of enzymes known as cellulases. The enzymatic mechanism of cellulases is well described, but the role of non-enzymatic components of the cellulose-degradation machinery is still poorly understood, and difficult to measure using experiments alone. In this study, we use a comprehensive set of atomistic molecular dynamics simulations to probe the molecular details of binding of the family-3a carbohydrate-binding module (CBM3a) and the bacterial expansin protein (EXLX1) to a range of cellulose substrates. Our results suggest that CBM3a behaves in a similar way on both crystalline and amorphous cellulose, whereas binding of the dual-domain expansin protein depends on the substrate crystallinity, and we relate our computed binding modes to the experimentally measured features of CBM and expansin action on cellulose.

The conversion of cellulosic biomass into biofuels requires degradation of the biomass into fermentable sugars. The most efficient natural cellulase system for carrying out this conversion is an extracellular multi-enzymatic complex named the cellulosome. In addition to temperature and pH stability, mechanical stability is important for functioning of cellulosome domains, and experimental techniques such as Single Molecule Force Spectroscopy (SMFS) have been used to measure the mechanical strength of several cellulosomal proteins. Molecular dynamics computer simulations provide complementary atomic-resolution quantitative maps of domain mechanical stability for identification of experimental leads for protein stabilization. In this study, we used multi-scale steered molecular dynamics computer simulations, benchmarked against new SMFS measurements, to measure the intermolecular contacts that confer high mechanical stability to a family 3 Carbohydrate Binding Module protein (CBM3) derived from the archetypal Clostridium thermocellum cellulosome. Our data predicts that electrostatic interactions in the calcium binding pocket modulate the mechanostability of the cellulose-binding module, which provides an additional design rule for the rational re-engineering of designer cellulosomes for biotechnology. Our data offers new molecular insights into the origins of mechanostability in cellulose binding domains and gives leads for synthesis of more robust cellulose-binding protein modules. On the other hand, simulations predict that insertion of a flexible strand can promote alternative unfolding pathways and dramatically reduce the mechanostability of the carbohydrate binding module, which gives routes to rational design of tailormade fingerprint complexes for force spectroscopy experiments.

Integrins are major players in cell adhesion and migration, and malfunctions in controlling their activity are associated with various diseases. Nevertheless, the details of integrin activation are not completely understood, and the role of lipids in the process is largely unknown. Herein, we show using atomistic molecular dynamics simulations that the interplay of phosphatidylinositol 4,5-bisphosphate (PIP2) and talin may directly alter the conformation of integrin αIIbβ3. Our results provide a new perspective on the role of PIP2 in integrin activation and indicate that the charged PIP2 lipid headgroup can perturb a clasp at the cytoplasmic face of the integrin heterodimer.

Phosphatidylinositol-transfer proteins (PITPs) regulate phosphoinositide signaling in eukaryotic cells. The defining feature of PITPs is their ability to exchange phosphatidylinositol (PtdIns) molecules between membranes, and this property is central to PITP-mediated regulation of lipid signaling. However, the details of the PITP-mediated lipid exchange cycle remain entirely obscure. Here, all-atom molecular dynamics simulations of the mammalian StART-like PtdIns/phosphatidylcholine (PtdCho) transfer protein PITP alpha, both on membrane bilayers and in solvated systems, informed downstream biochemical analyses that tested key aspects of the hypotheses generated by the molecular dynamics simulations. These studies provided five key insights into the PITP alpha lipid exchange cycle: (i) interaction of PITP alpha with the membrane is spontaneous and mediated by four specific protein substructures; (ii) the ability of PITP alpha to initiate closure around the PtdCho ligand is accompanied by loss of flexibility of two helix/loop regions, as well as of the C-terminal helix; (iii) the energy barrier of phospholipid extraction from the membrane is lowered by a network of hydrogen bonds between the lipid molecule and PITP alpha; (iv) the trajectory of PtdIns or PtdCho into and through the lipidbinding pocket is chaperoned by sets of PITP alpha residues conserved throughout the StART-like PITP family; and (v) conformational transitions in the C-terminal helix have specific functional involvements in PtdIns transfer activity. Taken together, these findings provide the first mechanistic description of key aspects of the PITP alpha PtdIns/PtdCho exchange cycle and offer a rationale for the high conservation of particular sets of residues across evolutionarily distant members of the metazoan StART-like PITP family.

Driven by interactions between lipids and proteins, biological membranes display lateral heterogeneity that manifests itself in a mosaic of liquid-ordered (Lo) or raft, and liquid-disordered (Ld) or non-raft domains with a wide range of different properties and compositions. In giant plasma membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB) to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1), whose headgroup accessibility and membrane order or phase partitioning may differ from those of GM1, rendering the interpretation of CTxB binding data quite problematic. To unravel the molecular basis of CTxB binding to GM1 and bdGM1, we explored the partitioning and the headgroup presentation of these gangliosides in the Lo and Ld phases using atomistic molecular dynamics simulations complemented by CTxB binding experiments. The conformation of both GM1 and bdGM1 was shown to be largely similar in the Lo and Ld phases. However, bdGM1 showed reduction in receptor availability when reconstituted into synthetic bilayer mixtures, highlighting that membrane phase partitioning of the gangliosides plays a considerable role in CTxB binding. Our results suggest that the CTxB binding is predominately modulated by the partitioning of the receptor to an appropriate membrane phase. Further, given that the Lo and Ld partitioning of bdGM1 differs from those of GM1, usage of bdGM1 for studying GM1 behavior in cells can lead to invalid interpretation of experimental data. publishedVersion

Transformation of cellulose into monosaccharides can be achieved in a chemical process performed by a special group of enzymes known as cellulases. We have used atomistic molecular dynamics simulations to study endoglucanase II (Cel5A) that is one of the proteins in this group. Based on the atomistic simulation results, we discuss how the Cel5A enzyme interacts with cellulose fibrils comprised of both crystalline and amorphous regions. We show that the enzyme’s carbohydrate-binding domain prefers to interact with crystalline regions of cellulose, while the catalytic domain has a high affinity to the amorphous regions of fibrils. In particular, through electrostatic interactions the catalytic domain attracts loose glucose chains to its catalytic cleft. The atomistic details of the enzyme–cellulose interaction are presented and the implications for practical applications are briefly discussed.

The epidermal growth factor receptor (EGFR) regulates several critical cellular processes and is an important target for cancer therapy. In lieu of a crystallographic structure of the complete receptor, atomistic molecular dynamics (MD) simulations have recently shown that they can excel in studies of the full-length receptor. Here we present atomistic MD simulations of the monomeric N-glycosylated human EGFR in biomimetic lipid bilayers that are, in parallel, also used for the reconstitution of full-length receptors. This combination enabled us to experimentally validate our simulations, using ligand binding assays and antibodies to monitor the conformational properties of the receptor reconstituted into membranes. We find that N-glycosylation is a critical determinant of EGFR conformation, and specifically the orientation of the EGFR ectodomain relative to the membrane. In the absence of a structure for full-length, posttranslationally modified membrane receptors, our approach offers new means to structurally define and experimentally validate functional properties of cell surface receptors in biomimetic membrane environments.

The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating an extraordinary discrimination of lipids sorted into these microvesicles. In particular, exosomes are highly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (mol% of total lipids). Furthermore, lipid species, even of classes not enriched in exosomes, were selectively included in exosomes. Finally, it was found that there is an 8.4-fold enrichment of lipids per mg of protein in exosomes. The detailed lipid composition provided in this study may be useful to understand the mechanism of exosome formation, release and function. Several of the lipids enriched in exosomes could potentially be used as cancer biomarkers.

Glycosylation where glycans are enzymatically attached to proteins is a common post-translational modification among many membrane protein families. The glycans associated with glycosylation serve a variety of structural and functional roles in protein structures, such as recognition and cell-cell adhesion. Determination of glycosylation in membrane protein structures is a complicated matter, though, since due to methodological limitations and the soft nature of glycans, the structure of the crystallized membrane proteins does not include the information arising from glycosylation. Here we use extensive atomistic molecular dynamics simulations together with experimental ligand binding assays and antibodies (1) to show for the N-glycosylated human epidermal growth factor receptor (EGFR) in biomimetic lipid bilayers that N-glycosylation is a crucial determinant of EGFR conformation, and specifically the orientation of the EGFR ectodomain relative to the membrane. It is expected that similar conclusions as to the importance of glycosylation in protein conformational behavior can be drawn for a number of other membrane proteins, too.(1) K. Kaszuba et al., Proc. Natl. Acad. Sci. USA 112, 4334 (2015).

A Sec14-nodulin protein model is used to identify the nodulin domain as a novel phosphoinositide effector module with a role in controlling lateral organization of phosphoinositide. The domain organization of Sec14-nodulin proteins suggests a versatile principle for the bit mapping of membrane surfaces into high-definition lipid-signaling screens., Polarized membrane morphogenesis is a fundamental activity of eukaryotic cells. This process is essential for the biology of cells and tissues, and its execution demands exquisite temporal coordination of functionally diverse membrane signaling reactions with high spatial resolution. Moreover, mechanisms must exist to establish and preserve such organization in the face of randomizing forces that would diffuse it. Here we identify the conserved AtSfh1 Sec14-nodulin protein as a novel effector of phosphoinositide signaling in the extreme polarized membrane growth program exhibited by growing Arabidopsis root hairs. The data are consistent with Sec14-nodulin proteins controlling the lateral organization of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) landmarks for polarized membrane morphogenesis in plants. This patterning activity requires both the PtdIns(4,5)P2 binding and homo-oligomerization activities of the AtSfh1 nodulin domain and is an essential aspect of the polarity signaling program in root hairs. Finally, the data suggest a general principle for how the phosphoinositide signaling landscape is physically bit mapped so that eukaryotic cells are able to convert a membrane surface into a high-definition lipid-signaling screen.