The FKBP-rapamycin binding domain of human TOR undergoes strong conformational changes in the presence of membrane mimetics with and without the regulator phosphatidic acid

The Ser/Thr kinase target of rapamycin (TOR) is a central controller of cellular growth and metabolism. Misregulation of TOR signaling is involved in metabolic and neurological disorders and tumor formation. TOR can be inhibited by association of a complex of rapamycin and FKBP12 to the FKBP12-rapamycin binding (FRB) domain. This domain was further proposed to interact with phosphatidic acid (PA), a lipid second messenger present in cellular membranes. Because mammalian TOR has been localized at various cellular membranes and in the nucleus, the output of TOR signaling may depend on its localization, which is expected to be influenced by the interaction with complex partners and regulators in response to cellular signals. Here, we present a detailed characterization of the interaction of the FRB domain with PA and how it is influenced by the surrounding membrane environment. On the basis of nuclear magnetic resonance- and circular dichroism-monitored binding studies using different neutral and negatively charged lipids as well as different membrane mimetics (micelles, bicelles, and liposomes), the FRB domain may function as a conditional peripheral membrane protein. However, the data for the isolated domain just indicate an increased affinity for negatively charged lipids and membrane patches but no specific preference for PA or PA-enriched regions. The membrane-mimetic environment induces strong conformational changes that largely maintain the α-helical secondary structure content but presumably disperse the helices in the lipidic environment. Consistent with overlapping binding surfaces for different lipids and the FKBP12-rapamycin complex, binding of the inhibitor complex protects the FRB domain from interactions with membrane mimetics at lower lipid concentrations.     

Emerging common themes in regulation of PIKKs and PI3Ks

Phosphatidylinositol‐3 kinase‐related kinases (PIKKs) comprise a family of protein kinases that respond to various stresses, including DNA damage, blocks in DNA replication, availability of nutrients and errors in mRNA splicing. PIKKs are characterized by the presence of a conserved kinase domain (KD), whose activity is regulated by two C‐terminal regions, referred to as PIKK‐regulatory domain (PRD) and FRAP‐ATM‐TRRAP‐C‐terminal (FATC), respectively. Here, we review functional and structural data that implicate the PRD and FATC domains in regulation of PIKK activity, drawing parallels to phosphatidylinositol‐3 kinases (PI3K), lipid kinases that have sequence similarity to PIKKs. The PI3K C‐terminus, which we propose to be equivalent to the PRD and FATC domains of PIKKs, is in close proximity to the activation loop of the KD, suggesting that in PIKKs, the PRD and FATC domains may regulate kinase activity by targeting the activation loop.     

Distant N- and C-terminal domains are required for intrinsic kinase activity of SMG-1, a critical component of nonsense-mediated mRNA decay

Phosphatidylinositol 3-kinase-related kinases (PIKKs) consisting of SMG-1, ATM, ATR, DNA-PKcs, and mTOR are a family of proteins involved in the surveillance of gene expression in eukaryotic cells. They are involved in mechanisms responsible for genome stability, mRNA quality, and translation. They share a large N-terminal domain and a C-terminal FATC domain in addition to the unique serine/threonine protein kinase (PIKK) domain that is different from classical protein kinases. However, structure-function relationships of PIKKs remain unclear. Here we have focused on one of the PIKK members, SMG-1, which is involved in RNA surveillance, termed nonsense-mediated mRNA decay (NMD), to analyze the roles of conserved and SMG-1-specific sequences on the intrinsic kinase activity. Analyses of sets of point and deletion mutants of SMG-1 in a purified system and intact cells revealed that the long N-terminal region and the conserved leucine in the FATC domain were essential for SMG-1 kinase activity. However, the conserved tryptophan in the TOR SMG-1 (TS) homology domain and the FATC domain was not. In addition, the long insertion region between PIKK and FATC domains was not essential for SMG-1 kinase activity. These results indicated an unexpected feature of SMG-1, i.e. that distantly located N- and C-terminal sequences were essential for the intrinsic kinase activity.     

A Highlights from MBoC Selection: Lipid domain–dependent regulation of single-cell wound repair

After damage, cells reseal their plasma membrane and repair the underlying cortical cytoskeleton. Although many different proteins have been implicated in cell repair, the potential role of specific lipids has not been explored. Here we report that cell damage elicits rapid formation of spatially organized lipid domains around the damage site, with different lipids concentrated in different domains as a result of both de novo synthesis and transport. One of these lipids—diacylglycerol (DAG)—rapidly accumulates in a broad domain that overlaps the zones of active Rho and Cdc42, GTPases that regulate repair of the cortical cytoskeleton. Formation of the DAG domain is required for Cdc42 and Rho activation and healing. Two DAG targets, protein kinase C (PKC) β and η, are recruited to cell wounds and play mutually antagonistic roles in the healing process: PKCβ participates in Rho and Cdc42 activation, whereas PKCη inhibits Rho and Cdc42 activation. The results reveal an unexpected diversity in subcellular lipid domains and the importance of such domains for a basic cellular process.     

The solution structure of the FATC domain of the protein kinase target of rapamycin suggests a role for redox-dependent structural and cellular stability

The target of rapamycin (TOR) is a highly conserved Ser/Thr kinase that plays a central role in the control of cellular growth. TOR has a characteristic multidomain structure. Only the kinase domain has catalytic function; the other domains are assumed to mediate interactions with TOR substrates and regulators. Except for the rapamycin-binding domain, there are no high-resolution structural data available for TOR. Here, we present a structural, biophysical, and mutagenesis study of the extremely conserved COOH-terminal FATC domain. The importance of this domain for TOR function has been highlighted in several publications. We show that the FATC domain, in its oxidized form, exhibits a novel structural motif consisting of an alpha-helix and a COOH-terminal disulfide-bonded loop between two completely conserved cysteine residues. Upon reduction, the flexibility of the loop region increases dramatically. The structural data, the redox potential of the disulfide bridge, and the biochemical data of a cysteine to serine mutant indicate that the intracellular redox potential can affect the cellular amount of the TOR protein via the FATC domain. Because the amount of TOR mRNA is not changed, the redox state of the FATC disulfide bond is probably influencing the degradation of TOR.     

Multiscale Simulations Suggest a Mechanism for the Association of the Dok7 PH Domain with PIP-Containing Membranes

Dok7 is a peripheral membrane protein that is associated with the MuSK receptor tyrosine kinase. Formation of the Dok7/MuSK/membrane complex is required for the activation of MuSK. This is a key step in the complex exchange of signals between neuron and muscle, which lead to neuromuscular junction formation, dysfunction of which is associated with congenital myasthenic syndromes. The Dok7 structure consists of a Pleckstrin Homology (PH) domain and a Phosphotyrosine Binding (PTB) domain. The mechanism of the Dok7 association with the membrane remains largely unknown. Using multi-scale molecular dynamics simulations we have explored the formation of the Dok7 PH/membrane complex. Our simulations indicate that the PH domain of Dok7 associates with membranes containing phosphatidylinositol phosphates (PIPs) via interactions of the β1/β2, β3/β4, and β5/β6 loops, which together form a positively charged surface on the PH domain and interact with the negatively charged headgroups of PIP molecules. The initial encounter of the Dok7 PH domain is followed by formation of additional interactions with the lipid bilayer, and especially with PIP molecules, which stabilizes the Dok7 PH/membrane complex. We have quantified the binding of the PH domain to the model bilayers by calculating a density landscape for protein/membrane interactions. Detailed analysis of the PH/PIP interactions reveal both a canonical and an atypical site to be occupied by the anionic lipid. PH domain binding leads to local clustering of PIP molecules in the bilayer. Association of the Dok7 PH domain with PIP lipids is therefore seen as a key step in localization of Dok7 to the membrane and formation of a complex with MuSK.     

The interplay between lipids and dopamine on α-synuclein oligomerization and membrane binding

The deposition of α-syn (α-synuclein) as amyloid fibrils and the selective loss of DA (dopamine) containing neurons in the substantia nigra are two key features of PD (Parkinson''s disease). α-syn is a natively unfolded protein and adopts an α-helical conformation upon binding to lipid membrane. Oligomeric species of α-syn have been proposed to be the pathogenic species associated with PD because they can bind lipid membranes and disrupt membrane integrity. DA is readily oxidized to generate reactive intermediates and ROS (reactive oxygen species) and in the presence of DA, α-syn form of SDS-resistant soluble oligomers. It is postulated that the formation of the α-syn:DA oligomers involves the cross-linking of DA-melanin with α-syn, via covalent linkage, hydrogen and hydrophobic interactions. We investigate the effect of lipids on DA-induced α-syn oligomerization and studied the ability of α-syn:DA oligomers to interact with lipids vesicles. Our results show that the interaction of α-syn with lipids inhibits the formation of DA-induced α-syn oligomers. Moreover, the α-syn:DA oligomer cannot interact with lipid vesicles or cause membrane permeability. Thus, the formation of α-syn:DA oligomers may alter the actions of α-syn which require membrane association, leading to disruption of its normal cellular function.     

NMR Characterization and Membrane Interactions of the Loop Region of Kindlin-3 F1 Subdomain

Kindlins-1,2 and 3 are FERM domain-containing cytosolic proteins involved in the activation and regulation of integrin-mediated cell adhesion. Apart from binding to integrin β cytosolic tails, kindlins and the well characterized integrin-activator talin bind membrane phospholipids. The ubiquitin-like F1 sub-domain of the FERM domain of talin contains a short loop that binds to the lipid membrane. By contrast, the F1 sub-domain of kindlins contains a long loop demonstrated binding to the membrane. Here, we report structural characterization and lipid interactions of the 83-residue F1 loop of kindlin-3 using NMR and optical spectroscopy methods. NMR studies demonstrated that the F1 loop of kindlin-3 is globally unfolded but stretches of residues assuming transient helical conformations could be detected in aqueous solution. We mapped membrane binding interactions of the F1 loop with small unilamellar vesicles (SUVs) containing either zwitterionic lipids or negatively charged lipids using ¹⁵N-¹H HSQC titrations. These experiments revealed that the F1 loop of kindlin-3 preferentially interacted with the negatively charged SUVs employing almost all of the residues. By contrast, only fewer residues appeared to be interacted with SUVs containing neutral lipids. Further, CD and NMR data suggested stabilization of helical conformations and predominant resonance perturbations of the F1 loop in detergent containing solutions. Conformations of an isolated N-terminal peptide fragment, or EK21, of the F1 loop, containing a poly-Lys sequence motif, important for membrane interactions, were also investigated in detergent solutions. EK21 adopted a rather extended or β-type conformations in complex with negatively charged SDS micelles. To our knowledge, this is the first report describing the conformations and residue-specific interactions of kindlin F1 loop with lipids. These data therefore provide important insights into the interactions of kindlin FERM domain with membrane lipids that contribute toward the integrin activating property.     

Characterization of residue-dependent differences in the peripheral membrane association of the FATC domain of the kinase ‘target of rapamycin’ by NMR and CD spectroscopy

The conserved C-terminal FATC domain of the kinase ‘target of rapamycin’ is important for its regulation and was suggested to contain a peripheral membrane anchor. Here, we present the characterization of the interactions of the yeast TOR1 FATC domain (2438–2470 = y1fatc) and 15 mutants with membrane mimetic micelles, bicelles, and small unilamellar vesicles (SUVs) by NMR and CD spectroscopy. Replacement of up to 6–7 residues did not result in a significant abrogation of the association with micelles or bicelles. However, replacement of only one residue could result in an impairment of the interaction with SUVs that are usually used at low concentrations. Some mutants not binding liposomes may be introduced in full-length TOR for future functional and localization studies in vivo.     

The C-Terminal V5 Domain of Protein Kinase Cα Is Intrinsically Disordered,with Propensity to Associate with a Membrane Mimetic

The C-terminal V5 domain is one of the most variable domains in Protein Kinase C isoforms (PKCs). V5 confers isoform specificity on its parent enzyme through interactions with isoform-specific adaptor proteins and possibly through specific intra-molecular interactions with other PKC domains. The structural information about V5 domains in solution is sparse. The objective of this work was to determine the conformational preferences of the V5 domain from the α isoform of PKC (V5α) and evaluate its ability to associate with membrane mimetics. We show that V5α and its phosphorylation-mimicking variant, dmV5α, are intrinsically disordered protein domains. Phosphorylation-mimicking mutations do not alter the overall conformation of the polypeptide backbone, as evidenced by the local nature of chemical shift perturbations and the secondary structure propensity scores. However, the population of the “cis-trans” conformer of the Thr⁶³⁸-Pro⁶³⁹-Pro⁶⁴⁰ turn motif, which has been implicated in the down-regulation of PKCα via peptidyl-prolyl isomerase Pin1, increases in dmV5α, along with the conformational flexibility of the region between the turn and hydrophobic motifs. Both wild type and dmV5α associate with micelles made of a zwitterionic detergent, n-dodecylphosphocholine. Upon micelle binding, V5α acquires a higher propensity to form helical structures at the conserved “NFD” motif and the entire C-terminal third of the domain. The ability of V5α to partition into the hydrophobic micellar environment suggests that it may serve as a membrane anchor during the PKC maturation process.     

α-Synuclein Senses Lipid Packing Defects and Induces Lateral Expansion of Lipids Leading to Membrane Remodeling

There is increasing evidence for the involvement of lipid membranes in both the functional and pathological properties of α-synuclein (α-Syn). Despite many investigations to characterize the binding of α-Syn to membranes, there is still a lack of understanding of the binding mode linking the properties of lipid membranes to α-Syn insertion into these dynamic structures. Using a combination of an optical biosensing technique and in situ atomic force microscopy, we show that the binding strength of α-Syn is related to the specificity of the lipid environment (the lipid chemistry and steric properties within a bilayer structure) and to the ability of the membranes to accommodate and remodel upon the interaction of α-Syn with lipid membranes. We show that this interaction results in the insertion of α-Syn into the region of the headgroups, inducing a lateral expansion of lipid molecules that can progress to further bilayer remodeling, such as membrane thinning and expansion of lipids out of the membrane plane. We provide new insights into the affinity of α-Syn for lipid packing defects found in vesicles of high curvature and in planar membranes with cone-shaped lipids and suggest a comprehensive model of the interaction between α-Syn and lipid bilayers. The ability of α-Syn to sense lipid packing defects and to remodel membrane structure supports its proposed role in vesicle trafficking.     

A Novel Structural Unit in the N-terminal Region of Filamins

Immunoglobulin-like (Ig) domains are a widely expanded superfamily that act as interaction motifs or as structural spacers in multidomain proteins. Vertebrate filamins (FLNs), which are multifunctional actin-binding proteins, consist of 24 Ig domains. We have recently discovered that in the C-terminal rod 2 region of FLN, Ig domains interact with each other forming functional domain pairs, where the interaction with signaling and transmembrane proteins is mechanically regulated by weak actomyosin contraction forces. Here, we investigated if there are similar inter-domain interactions around domain 4 in the N-terminal rod 1 region of FLN. Protein crystal structures revealed a new type of domain organization between domains 3, 4, and 5. In this module, domains 4 and 5 interact rather tightly, whereas domain 3 has a partially flexible interface with domain 4. NMR peptide titration experiments showed that within the three-domain module, domain 4 is capable for interaction with a peptide derived from platelet glycoprotein Ib. Crystal structures of FLN domains 4 and 5 in complex with the peptide revealed a typical β sheet augmentation interaction observed for many FLN ligands. Domain 5 was found to stabilize domain 4, and this could provide a mechanism for the regulation of domain 4 interactions.     

Annular Anionic Lipids Stabilize the Integrin αIIbβ3 Transmembrane Complex

Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin αIIbβ3 transmembrane (TM) complex is stabilized by a membrane-proximal αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for αIIb(Arg⁹⁹⁵) contacts with β3(Asp⁷²³) but paradoxically do not diminish the contribution of αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) to TM complex stability. Overall, anionic lipids in annular positions stabilize the αIIbβ3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact.     

Surface Tensiometry of Apolipoprotein B Domains at Lipid Interfaces Suggests a New Model for the Initial Steps in Triglyceride-rich Lipoprotein Assembly

Apolipoprotein B (apoB) is the principal protein component of triacylglyceride (TAG)-rich lipoproteins, including chylomicrons and very low density lipoprotein, which is the precursor to LDL (the “bad cholesterol”). TAG-rich lipoprotein assembly is initiated by the N-terminal βα1 superdomain of apoB, which co-translationally binds and remodels the luminal leaflet of the rough endoplasmic reticulum. The βα1 superdomain contains four domains and is predicted to interact directly with lipids. Using drop tensiometry, we examined the interfacial properties of the α-helical and C-sheet domains and several subdomains to establish a detailed structure-function relationship at the lipid/water interface. The adsorption, stress response, exchangeability, and pressure (Π)-area relationship were studied at both triolein/water and triolein/1-palmitoyl, 2-oleoylphosphatidylcholine/water interfaces that mimic physiological environments. The α-helical domain spontaneously adsorbed to a triolein/water interface and formed a viscoelastic surface. It was anchored to the surface by helix 6, and the other helices were ejected and/or remodeled on the surface as a function of surface pressure. The C-sheet instead formed an elastic film on a triolein/water interface and was irreversibly anchored to the lipid surface, which is consistent with the behavior of amphipathic β-strands. When both domains were adsorbed together on the surface, the C-sheet shielded a portion of the α-helical domain from the surface, which retained its globular structure. Overall, the unique secondary and tertiary structures of the N-terminal domains of apoB support the intrinsic capability of co-translational lipid recruitment. The evidence presented here allows the construction of a detailed model of the initiation of TAG-rich lipoprotein assembly.     

Partition profile of the nicotinic acetylcholine receptor in lipid domains upon reconstitution

The nicotinic acetylcholine receptor (AChR) is in intimate contact with the lipids in its native membrane. Here we analyze the possibility that it is the intrinsic properties of the AChR that determine its partition into a given lipid domain. Torpedo AChR or a synthetic peptide corresponding to the AChR γM4 segment (the one in closer contact with lipids) was reconstituted into “raft”-containing model membranes. The distribution of the AChR was assessed by Triton X-100 extraction in combination with fluorescence studies, and lipid analyses were performed on each sample. The influence of rapsyn, a peripheral protein involved in AChR aggregation, was studied. Raft-like domain aggregation was also studied using membranes containing the ganglioside GM1 followed by GM1 crosslinking. The γM4 peptide displays a marked preference for raft-like domains. In contrast, AChR alone or in the presence of rapsyn or ganglioside aggregation exhibits no such preference for raft-like domains, but it does cause a significant reduction in the total amount of these domains. The results indicate that the distribution of the AChR in lipid domains cannot be due exclusively to the intrinsic physicochemical properties of the protein and that there must be an external signal in native cell membranes that directs the AChR to a specific membrane domain.     

Solution Structure of Human Growth Arrest and DNA Damage 45α (Gadd45α) and Its Interactions with Proliferating Cell Nuclear Antigen (PCNA) and Aurora A Kinase

Gadd45α is a nuclear protein encoded by a DNA damage-inducible gene. Through its interactions with other proteins, Gadd45α participates in the regulation of DNA repair, cell cycle, cell proliferation, and apoptosis. The NMR structure of human Gadd45α has been determined and shows an α/β fold with two long disordered and flexible regions at the N terminus and one of the loops. Human Gadd45α is predominantly monomeric in solution but exists in equilibrium with dimers and other oligomers whose population increases with protein concentration. NMR analysis shows that Aurora A interacts through its N-terminal domain with a region of human Gadd45α encompassing the site of dimerization, suggesting that the oligomerization of Gadd45α could be a regulatory mechanism to modulate its interactions with Aurora A, and possibly with other proteins too. However, Gadd45α appears to interact only weakly with PCNA through its flexible loop, in contrast with previous and contradictory reports.     

Importance of Protein-tyrosine Phosphatase-α Catalytic Domains for Interactions with SHP-2 and Interleukin-1-induced Matrix Metalloproteinase-3 Expression

Interleukin-1 (IL-1) induces extracellular matrix degradation as a result of increased expression of matrix metalloproteinases (MMPs). We examined adhesion-restricted signaling pathways that enable IL-1-induced MMP release in human gingival and murine fibroblasts. Of the seven MMPs and three tissue inhibitors of MMPs screened, IL-1 enhanced release only of MMP3 when cells formed focal adhesions. Inhibition of protein-tyrosine phosphatases (PTPs), which are enriched in focal adhesions, blocked IL-1-induced MMP3 release. Accordingly, in contrast to wild-type cells, fibroblasts null for PTPα did not exhibit IL-1-induced MMP3 release. IL-1 treatment enhanced the recruitment of SHP-2 and PTPα to focal adhesions and the association of PTPα with SHP-2. Pulldown assays confirmed a direct interaction between PTPα and SHP-2, which was dependent on the intact, membrane-proximal phosphatase domain of PTPα. Interactions between SHP-2 and PTPα, recruitment of SHP-2 to focal adhesions, IL-1-induced ERK activation, and MMP3 expression were all blocked by point mutations in the phosphatase domains of PTPα. These data indicate that IL-1-induced signaling through focal adhesions leading to MMP3 release and interactions between SHP-2 and PTPα are dependent on the integrity of the catalytic domains of PTPα.     

A Role for the Disintegrin Domain of Cyritestin, a Sperm Surface Protein Belonging to the ADAM Family, in Mouse Sperm–Egg Plasma Membrane Adhesion and Fusion

Sperm–egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell–cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm–egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (α and β subunits). Fertilin α and β are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin β functions in sperm–egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin α, fertilin β, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin β, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm–egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80–90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin β active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin β also strongly inhibited (80–90%) both sperm–egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin β showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin β in sperm–egg plasma membrane adhesion leading to fusion.     

Dynamics of the Antigen-binding Grooves in CD1 Proteins: REVERSIBLE HYDROPHOBIC COLLAPSE IN THE LIPID-FREE STATE*

CD1 proteins mediate the presentation of endogenous and foreign lipids on the cell surface for recognition by T cell receptors. To sample a diverse antigen pool, CD1 proteins are repeatedly internalized and recycled, assisted, in some cases, by lipid transfer proteins such as saposins. The specificity of each CD1 isoform is, therefore, conferred in part by its intracellular pathway but also by distinct structural features of the antigen-binding domain. Crystal structures of CD1-lipid complexes reveal hydrophobic grooves and pockets within these binding domains that appear to be specialized for different lipids. However, the mechanism of lipid loading and release remains to be characterized. Here we gain insights into this mechanism through a meta-analysis of the five human CD1 isoforms, in the lipid-bound and lipid-free states, using all-atom molecular dynamics simulations. Strikingly, for isoforms CD1b through CD1e, our simulations show the near-complete collapse of the hydrophobic cavities in the absence of the antigen. This event results from the spontaneous closure of the binding domain entrance, flanked by two α-helices. Accordingly, we show that the anatomy of the binding cavities is restored if these α-helices are repositioned extrinsically, suggesting that helper proteins encountered during recycling facilitate lipid exchange allosterically. By contrast, we show that the binding cavity of CD1a is largely preserved in the unliganded state because of persistent electrostatic interactions that keep the portal α-helices at a constant separation. The robustness of this binding groove is consistent with the observation that lipid exchange in CD1a is not dependent on cellular internalization.     

Heat Shock Protein 90 Associates with the Per-Arnt-Sim Domain of Heme-free Soluble Guanylate Cyclase: IMPLICATIONS FOR ENZYME MATURATION*

Heat shock protein 90 (hsp90) drives heme insertion into the β1 subunit of soluble guanylate cyclase (sGC) β1, which enables it to associate with a partner sGCα1 subunit and mature into a nitric oxide (NO)-responsive active form. We utilized fluorescence polarization measurements and hydrogen-deuterium exchange mass spectrometry to define molecular interactions between the specific human isoforms hsp90β and apo-sGCβ1. hsp90β and its isolated M domain, but not its isolated N and C domains, bind with low micromolar affinity to a heme-free, truncated version of sGCβ1 (sGCβ1(1–359)-H105F). Surprisingly, hsp90β and its M domain bound to the Per-Arnt-Sim (PAS) domain of apo-sGC-β1(1–359), which lies adjacent to its heme-binding (H-NOX) domain. The interaction specifically involved solvent-exposed regions in the hsp90β M domain that are largely distinct from sites utilized by other hsp90 clients. The interaction strongly protected two regions of the sGCβ1 PAS domain and caused local structural relaxation in other regions, including a PAS dimerization interface and a segment in the H-NOX domain. Our results suggest a means by which the hsp90β interaction could prevent apo-sGCβ1 from associating with its partner sGCα1 subunit while enabling structural changes to assist heme insertion into the H-NOX domain. This mechanism would parallel that in other clients like the aryl hydrocarbon receptor and HIF1α, which also interact with hsp90 through their PAS domains to control protein partner and small ligand binding interactions.