Structural insights into the aPKC regulatory switch mechanism of the human cell polarity protein lethal giant larvae 2.

Metazoan cell polarity is controlled by a set of highly conserved proteins. Lethal giant larvae (Lgl) functions in apical-basal polarity through phosphorylation-dependent interactions with several other proteins as well as the plasma membrane. Phosphorylation of Lgl by atypical protein kinase C (aPKC), a component of the partitioning-defective (Par) complex in epithelial cells, excludes Lgl from the apical membrane, a crucial step in the establishment of epithelial cell polarity. We present the crystal structures of human Lgl2 in both its unphosphorylated and aPKC-phosphorylated states. Lgl2 adopts a double ß-propeller structure that is unchanged by aPKC phosphorylation of an unstructured loop in its second ß-propeller, ruling out models of phosphorylation-dependent conformational change. We demonstrate that phosphorylation controls the direct binding of purified Lgl2 to negative phospholipids in vitro. We also show that a coil-helix transition of this region that is promoted by phosphatidylinositol 4,5-bisphosphate (PIP2) is also phosphorylation-dependent, implying a highly effective phosphorylative switch for membrane association.

Solving the structure of Lgl2, a difficult blind test of unsupervised structure determination.

In the companion paper by Ufimtsev and Levitt [Ufimtsev IS, Levitt M (2019) Proc Natl Acad Sci USA, 10.1073/pnas.1821512116], we presented a method for unsupervised solution of protein crystal structures and demonstrated its utility by solving several test cases of known structure in the 2.9- to 3.45-Å resolution range. Here we apply this method to solve the crystal structure of a 966-amino acid construct of human lethal giant larvae protein (Lgl2) that resisted years of structure determination efforts, at 3.2-Å resolution. The structure was determined starting with a molecular replacement (MR) model identified by unsupervised refinement of a pool of 50 candidate MR models. This initial model had 2.8-Å RMSD from the solution. The solved structure was validated by comparison with a model subsequently derived from an alternative crystal form diffracting to higher resolution. This model could phase an anomalous difference Fourier map from an Hg derivative, and a single-wavelength anomalous dispersion phased density map made from these sites aligned with the refined structure.

Dynamic distinctions in the Na+/Ca2+ exchanger adopting the inward- and outward-facing conformational states.

Na+/Ca2+ exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na+ or Ca2+ specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.

Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation.

Na(+)/Ca(2+) exchangers (NCXs) are ubiquitous membrane transporters with a key role in Ca(2+) homeostasis and signaling. NCXs mediate the bidirectional translocation of either Na(+) or Ca(2+), and thus can catalyze uphill Ca(2+) transport driven by a Na(+) gradient, or vice versa. In a major breakthrough, a prokaryotic NCX homolog (NCX_Mj) was recently isolated and its crystal structure determined at atomic resolution. The structure revealed an intriguing architecture consisting of two inverted-topology repeats, each comprising five transmembrane helices. These repeats adopt asymmetric conformations, yielding an outward-facing occluded state. The crystal structure also revealed four putative ion-binding sites, but the occupancy and specificity thereof could not be conclusively established. Here, we use molecular-dynamics simulations and free-energy calculations to identify the ion configuration that best corresponds to the crystallographic data and that is also thermodynamically optimal. In this most probable configuration, three Na(+) ions occupy the so-called Sext, SCa, and Sint sites, whereas the Smid site is occupied by one water molecule and one H(+), which protonates an adjacent aspartate side chain (D240). Experimental measurements of Na(+)/Ca(2+) and Ca(2+)/Ca(2+) exchange by wild-type and mutagenized NCX_Mj confirm that transport of both Na(+) and Ca(2+) requires protonation of D240, and that this side chain does not coordinate either ion at Smid. These results imply that the ion exchange stoichiometry of NCX_Mj is 3:1 and that translocation of Na(+) across the membrane is electrogenic, whereas transport of Ca(2+) is not. Altogether, these findings provide the basis for further experimental and computational studies of the conformational mechanism of this exchanger.

Competitive and non-competitive regulation of calcium-dependent inactivation in CaV1.2 L-type Ca2+ channels by calmodulin and Ca2+-binding protein 1.

CaV1.2 interacts with the Ca(2+) sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, α1C. Ca(2+)/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca(2+) ions (Ca(2+)-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the α1C- subunit using Förster resonance energy transfer (FRET) and recording of Cav1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of α1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for α1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaM altered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15-20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated α1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that the NT is not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in α1C.

Structural flexibility of CaV1.2 and CaV2.2 I-II proximal linker fragments in solution.

Voltage-dependent calcium channels (CaV) enable the inward flow of calcium currents for a wide range of cells. CaV1 and CaV2 subtype α1 subunits form the conducting pore using four repeated membrane domains connected by intracellular linkers. The domain I-II linker connects to the membrane gate (IS6), forming an α-helix, and is bound to the CaVß subunit. Previous studies indicated that this region may or may not form a continuous helix depending on the CaV subtype, thereby modulating channel activation and inactivation properties. Here, we used small-angle x-ray scattering and ensemble modeling analysis to investigate the solution structure of these linkers, extending from the membrane domain and including the CaVß-binding site, called the proximal linker (PL). The results demonstrate that the CaV1.2 PL is more flexible than the CaV2.2 PL, the flexibility is intrinsic and not dependent on CaVß binding, and the flexibility can be most easily explained by the presence of conserved glycines. Our analysis also provides a robust example of investigating protein domains in which flexibility plays an essential role.

The role of a voltage-dependent Ca2+ channel intracellular linker: a structure-function analysis.

Voltage-dependent calcium channels (VDCCs) allow the passage of Ca(2+) ions through cellular membranes in response to membrane depolarization. The channel pore-forming subunit, α1, and a regulatory subunit (Ca(V)ß) form a high affinity complex where Ca(V)ß binds to a α1 interacting domain in the intracellular linker between α1 membrane domains I and II (I-II linker). We determined crystal structures of Ca(V)ß2 functional core in complex with the Ca(V)1.2 and Ca(V)2.2 I-II linkers to a resolution of 1.95 and 2.0 Å, respectively. Structural differences between the highly conserved linkers, important for coupling Ca(V)ß to the channel pore, guided mechanistic functional studies. Electrophysiological measurements point to the importance of differing linker structure in both Ca(V)1 and 2 subtypes with mutations affecting both voltage- and calcium-dependent inactivation and voltage dependence of activation. These linker effects persist in the absence of Ca(V)ß, pointing to the intrinsic role of the linker in VDCC function and suggesting that I-II linker structure can serve as a brake during inactivation.

Asymmetric Preorganization of Inverted Pair Residues in the Sodium-Calcium Exchanger.

In analogy with many other proteins, Na(+)/Ca(2+) exchangers (NCX) adapt an inverted twofold symmetry of repeated structural elements, while exhibiting a functional asymmetry by stabilizing an outward-facing conformation. Here, structure-based mutant analyses of the Methanococcus jannaschii Na(+)/Ca(2+) exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na(+)or Ca(2+) binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct "noncatalytic" residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins.

Interactions between N and C termini of α1C subunit regulate inactivation of CaV1.2 L-type Ca(2+) channel.

The modulation and regulation of voltage-gated Ca(2+) channels is affected by the pore-forming segments, the cytosolic parts of the channel, and interacting intracellular proteins. In this study we demonstrate a direct physical interaction between the N terminus (NT) and C terminus (CT) of the main subunit of the L-type Ca(2+) channel CaV1.2, α1C, and explore the importance of this interaction for the regulation of the channel. We used biochemistry to measure the strength of the interaction and to map the location of the interaction sites, and electrophysiology to investigate the functional impact of the interaction. We show that the full-length NT (amino acids 1-154) and the proximal (close to the plasma membrane) part of the CT, pCT (amino acids 1508-1669) interact with sub-micromolar to low-micromolar affinity. Calmodulin (CaM) is not essential for the binding. The results further suggest that the NT-CT interaction regulates the channel's inactivation, and that Ca(2+), presumably through binding to calmodulin (CaM), reduces the strength of NT-CT interaction. We propose a molecular mechanism in which NT and CT of the channel serve as levers whose movements regulate inactivation by promoting changes in the transmembrane core of the channel via S1 (NT) or S6 (pCT) segments of domains I and IV, accordingly, and not as a kind of pore blocker. We hypothesize that Ca(2+)-CaM-induced changes in NT-CT interaction may, in part, underlie the acceleration of CaV1.2 inactivation induced by Ca(2+) entry into the cell.

Functional asymmetry of bidirectional Ca2+-movements in an archaeal sodium-calcium exchanger (NCX_Mj).

Dynamic features of Ca(2+) interactions with transport and regulatory sites control the Ca(2+)-fluxes in mammalian Na(+)/Ca(2+)(NCX) exchangers bearing the Ca(2+)-binding regulatory domains on the cytosolic 5L6 loop. The crystal structure of Methanococcus jannaschii NCX (NCX_Mj) may serve as a template for studying ion-transport mechanisms since NCX_Mj does not contain the regulatory domains. The turnover rate of Na(+)/Ca(2+) exchange (kcat=0.5±0.2 s(-1)) in WT-NCX_Mj is 10(3)-10(4) times slower than in mammalian NCX. In NCX_Mj, the intrinsic equilibrium (Kint) for bidirectional Ca(2+) movements (defined as the ratio between the cytosolic and extracellular Km of Ca(2+)/Ca(2+) exchange) is asymmetric, Kint=0.15±0.5. Therefore, the Ca(2+) movement from the cytosol to the extracellular side is ∼7-times faster than in the opposite direction, thereby representing a stabilization of outward-facing (extracellular) access. This intrinsic asymmetry accounts for observed differences in the cytosolic and extracellulr Km values having a physiological relevance. Bidirectional Ca(2+) movements are also asymmetric in mammalian NCX. Thus, the stabilization of the outward-facing access along the transport cycle is a common feature among NCX orthologs despite huge differences in the ion-transport kinetics. Elongation of the cytosolic 5L6 loop in NCX_Mj by 8 or 14 residues accelerates the ion transport rates (kcat) ∼10 fold, while increasing the Kint values 100-250-fold (Kint=15-35). Therefore, 5L6 controls both the intrinsic equilibrium and rates of bidirectional Ca(2+) movements in NCX proteins. Some additional structural elements may shape the kinetic variances among phylogenetically distant NCX variants, although the intrinsic asymmetry (Kint) of bidirectional Ca(2+) movements seems to be comparable among evolutionary diverged NCX variants.

The C2B domain is the primary Ca2+ sensor in DOC2B: a structural and functional analysis.

DOC2B (double-C2 domain) protein is thought to be a high-affinity Ca(2+) sensor for spontaneous and asynchronous neurotransmitter release. To elucidate the molecular features underlying its physiological role, we determined the crystal structures of its isolated C2A and C2B domains and examined their Ca(2+)-binding properties. We further characterized the solution structure of the tandem domains (C2AB) using small-angle X-ray scattering. In parallel, we tested structure-function correlates with live cell imaging tools. We found that, despite striking structural similarity, C2B binds Ca(2+) with considerably higher affinity than C2A. The C2AB solution structure is best modeled as two domains with a highly flexible orientation and no difference in the presence or absence of Ca(2+). In addition, kinetic studies of C2AB demonstrate that, in the presence of unilamellar vesicles, Ca(2+) binding is stabilized, as reflected by the ~10-fold slower rate of Ca(2+) dissociation than in the absence of vesicles. In cells, isolated C2B translocates to the plasma membrane (PM) with an EC50 of 400 nM while the C2A does not translocate at submicromolar Ca(2+) concentrations, supporting the biochemical observations. Nevertheless, C2AB translocates to the PM with an ~2-fold lower EC50 and to a greater extent than C2B. Our results, together with previous studies, reveal that the C2B is the primary Ca(2+) sensing unit in DOC2B, whereas C2A enhances the interaction of C2AB with the PM.

Ca(V)1.2 I-II linker structure and Timothy syndrome.

Ca(V) channels are multi-subunit protein complexes that enable inward cellular Ca(2+) currents in response to membrane depolarization. We recently described structure-function studies of the intracellular α1 subunit domain I-II linker, directly downstream of domain IS6. The results show the extent of the linker's helical structure to be subfamily dependent, as dictated by highly conserved primary sequence differences. Moreover, the difference in structure confers different biophysical properties, particularly the extent and kinetics of voltage and calcium-dependent inactivation. Timothy syndrome is a human genetic disorder due to mutations in the Ca(V)1.2 gene. Here, we explored whether perturbation of the I-II linker helical structure might provide a mechanistic explanation for a Timothy syndrome mutant's (human Ca(V)1.2 G406R equivalent) biophysical effects on inactivation and activation. The results are equivocal, suggesting that a full mechanistic explanation for this Timothy syndrome mutation requires further investigation.

Characterization of the calmodulin-binding site in the N terminus of CaV1.2.

Interaction of calmodulin (CaM) with the C-terminus (CT) of the L-type Ca(V)1.2 channel is crucial for Ca(2+)-dependent inactivation (CDI). CaM also binds to the N-terminus (NT), and a CaM-formed "bridge" between CT and NT has been proposed to control CDI. We characterized the interaction of CaM with its NT-binding peptide. Binding is Ca(2+)-dependent with an affinity of 0.6 microM. Mutations in NT of Ca(V)1.2 that abolished the binding of CaM only slightly weakened the CDI but also accelerated the VDI. CaM did not foster an interaction between the CaM-binding peptides of NT and CT. Thus, the role of CaM's interaction with the Ca(V)1.2 NT remains to be determined.