N-terminal Helical Domain Research Articles

Crystal structure of an engineered YopM-InlB hybrid protein

BackgroundThe multi-domain protein InlB (internalin B) from Listeria monocytogenes is an agonist of the human receptor tyrosine kinase MET. Only the internalin domain directly interacts with MET. The internalin domain consists of seven central leucine-rich repeats (LRRs) flanked by an N-terminal helical cap domain and a C-terminal immunoglobulin-like structure. A potential function of the N-terminal cap in receptor binding could so far not be demonstrated by deleting the cap, since the cap is also implicated in nucleating folding of the LRR domain.ResultsWe generated an InlB variant (YopM-InlB) in which the InlB cap domain was replaced by the unrelated N-terminal capping structure of the LRR protein YopM from Yersinia enterocolitica. The crystal structure of the engineered protein shows that it folds properly. Because the first LRR is structurally closely linked to the cap domain, we exchanged LRR1 along with the cap domain. This resulted in unexpected structural changes extending to LRR2 and LRR3, which are deeply involved in MET binding. As a consequence, the binding of YopM-InlB to MET was substantially weaker than that of wild type InlB. The engineered protein was about one order of magnitude less active in colony scatter assays than wild type InlB.ConclusionsWe obtained a well-behaved InlB variant with an altered N-terminal capping structure through protein design. The reduced affinity for MET precludes a straightforward interpretation of the results from cell-based assays. Still, the engineered hybrid protein induced cell scatter, suggesting that the cap is required for folding and stability of InlB but is not essential for interactions that assemble the signalling-active receptor complex. The cap swap approach described here is clearly applicable to other L. monocytogenes internalins and other LRR proteins such as YopM and may yield useful structure/function correlates within this protein family.

Structural Basis for Proteasome Formation Controlled by an Assembly Chaperone Nas2

Proteasome formation does not occur due to spontaneous self-organization but results from a highly ordered process assisted by several assembly chaperones. The assembly of the proteasome ATPase subunits is assisted by four client-specific chaperones, of which three have been structurally resolved. Here, we provide the structural basis for the working mechanisms of the last, hereto structurally uncharacterized assembly chaperone, Nas2. We revealed that Nas2 binds to the Rpt5 subunit in a bivalent mode: the N-terminal helical domain of Nas2 masks the Rpt1-interacting surface of Rpt5, whereas its C-terminal PDZ domain caps the C-terminal proteasome-activating motif. Thus, Nas2 operates as a proteasome activation blocker, offering a checkpoint during the formation of the 19S ATPase prior to its docking onto the proteolytic 20S core particle.

Malonyl-CoA decarboxylase deficiency: Long-term follow-up of a patient new clinical features and novel mutations

Malonyl-CoA decarboxylase (MLYCD, EC 4.1.1.9) deficiency is a rare autosomal recessive disorder that is widely diagnosed by neonatal screening. We report long term follow up of a patient with MLYCD deficiency showing signs of neonatal hypoglycemia, mental retardation, developmental delay and rheumatoid arthritis. Brain MRI revealed patchy, symmetrical hyperintensity of the deep white matter with periventricular white matter and subcortical arcuate fibers being spared. MLCYD gene sequence analysis was done to identify possible mutations. Expression analyses at mRNA and protein levels were also performed. Further, immunocytochemical studies were implemented to check for its subcellular localization. MLYCD gene sequencing identified a novel compound heterozygous mutation (c.22 T>A, p.M1K, c.454 C>A; pH152N) in our patient and a heterozygous mutation in the healthy mother c.22 T>A; pM1K. Reduced expression of RNA and protein levels was observed. Immunocytochemical analysis showed diffused staining across the cytoplasm with apparent signs of intracellular mislocalization to the nucleus. RESULTS also indicated subcellular colocalization of MLCYD with mitochondria was scant compared to control. Our patient was identified with a novel compound heterozygous MLYCD mutation at the N-terminal helical domain. This study indicates that protein mislocalization is a characteristic feature of MLYCD deficiency in our patient.

The TLR signalling adaptor TRIF/TICAM-1 has an N-terminal helical domain with structural similarity to IFIT proteins

TRIF/TICAM-1 (TIR domain-containing adaptor inducing interferon-β/TIR domain-containing adaptor molecule 1) is the adaptor protein in the Toll-like receptor (TLR) 3 and 4 signalling pathway that leads to the production of type 1 interferons and cytokines. The signalling involves TIR (Toll/interleukin-1 receptor) domain-dependent TRIF oligomerization. A protease-resistant N-terminal region is believed to be involved in self-regulation of TRIF by interacting with its TIR domain. Here, the structural and functional characterization of the N-terminal domain of TRIF (TRIF-NTD) comprising residues 1-153 is reported. The 2.22 Å resolution crystal structure was solved by single-wavelength anomalous diffraction (SAD) using selenomethionine-labelled crystals of TRIF-NTD containing two additional introduced Met residues (TRIF-NTDA66M/L113M). The structure consists of eight antiparallel helices that can be divided into two subdomains, and the overall fold shares similarity to the interferon-induced protein with tetratricopeptide repeats (IFIT) family of proteins, which are involved in both the recognition of viral RNA and modulation of innate immune signalling. Analysis of TRIF-NTD surface features and the mapping of sequence conservation onto the structure suggest several possible binding sites involved in either TRIF auto-regulation or interaction with other signalling molecules or ligands. TRIF-NTD suppresses TRIF-mediated activation of the interferon-β promoter, as well as NF-κB-dependent reporter-gene activity. These findings thus identify opportunities for the selective targeting of TLR3- and TLR4-mediated inflammation.

Crystal Structures of Malonyl-Coenzyme A Decarboxylase Provide Insights into Its Catalytic Mechanism and Disease-Causing Mutations

SummaryMalonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design.

Structural insights into ChpT, an essential dimeric histidine phosphotransferase regulating the cell cycle in Caulobacter crescentus.

Two-component and phosphorelay signal-transduction proteins are crucial for bacterial cell-cycle regulation in Caulobacter crescentus. ChpT is an essential histidine phosphotransferase that controls the activity of the master cell-cycle regulator CtrA by phosphorylation. Here, the 2.2 Å resolution crystal structure of ChpT is reported. ChpT is a homodimer and adopts the domain architecture of the intracellular part of class I histidine kinases. Each subunit consists of two distinct domains: an N-terminal helical hairpin domain and a C-terminal α/β domain. The two N-terminal domains are adjacent within the dimer, forming a four-helix bundle. The ChpT C-terminal domain adopts an atypical Bergerat ATP-binding fold.

Crystal structure of Arabidopsis cyclophilin38 reveals a previously uncharacterized immunophilin fold and a possible autoinhibitory mechanism.

Cyclophilin38 (CYP38) is one of the highly divergent cyclophilins from Arabidopsis thaliana. Here, we report the crystal structure of the At-CYP38 protein (residues 83 to 437 of 437 amino acids) at 2.39-Å resolution. The structure reveals two distinct domains: an N-terminal helical bundle and a C-terminal cyclophilin β-barrel, connected by an acidic loop. Two N-terminal β-strands become part of the C-terminal cyclophilin β-barrel, thereby making a previously undiscovered domain organization. This study shows that CYP38 does not possess peptidyl-prolyl cis/trans isomerase activity and identifies a possible interaction of CYP38 with the E-loop of chlorophyll protein47 (CP47), a component of photosystem II. The interaction of CYP38 with the E-loop of CP47 is mediated through its cyclophilin domain. The N-terminal helical domain is closely packed together with the putative C-terminal cyclophilin domain and establishes a strong intramolecular interaction, thereby preventing the access of the cyclophilin domain to other proteins. This was further verified by protein-protein interaction assays using the yeast two-hybrid system. Furthermore, the non-Leucine zipper N-terminal helical bundle contains several new elements for protein-protein interaction that may be of functional significance. Together, this study provides the structure of a plant cyclophilin and explains a possible mechanism for autoinhibition of its function through an intramolecular interaction.

Effect of the A30P mutation on the structural dynamics of micelle-bound αSynuclein released in water: a molecular dynamics study

Atomistic molecular dynamics simulation has been used to probe the effect of the A30P mutation on the structural dynamics of micelle-bound, helical αSynuclein when released in an aqueous environment. On the timescales simulated, the effect of the mutation on the secondary structure is restricted to local changes close to the mutation site in the N-terminal helical domain. The changes are transient, and all residues except Lys23 recover their initial structure. The local behavior due to the mutation gives rise to a global difference in the A30P mutant in the form of a permanent kink in the N-terminal helical domain.

Structure and Functional Characterization of the RNA-Binding Element of the NLRX1 Innate Immune Modulator

Mitochondrial NLRX1 is a member of the family of nucleotide-binding domain and leucine-rich-repeat-containing proteins (NLRs) that mediate host innate immunity as intracellular surveillance sensors against common molecular patterns of invading pathogens. NLRX1 functions in antiviral immunity, but the molecular mechanism of its ligand-induced activation is largely unknown. The crystal structure of the C-terminal fragment (residues 629-975) of human NLRX1 (cNLRX1) at 2.65 Å resolution reveals that cNLRX1 consists of an N-terminal helical (LRRNT) domain, central leucine-rich repeat modules (LRRM), and a C-terminal three-helix bundle (LRRCT). cNLRX1 assembles into a compact hexameric architecture that is stabilized by intersubunit and interdomain interactions of LRRNT and LRRCT in the trimer and dimer components of the hexamer, respectively. Furthermore, we find that cNLRX1 interacts directly with RNA and supports a role for NLRX1 in recognition of intracellular viral RNA in antiviral immunity.

The β-Catenin Binding Protein ICAT Modulates Androgen Receptor Activity

Androgens have important roles in the development of the prostate gland and in prostate cancer. Since the finding that β-catenin is a cofactor of the androgen receptor (AR) and can augment AR signaling, several proteins have been found to affect AR signaling through their interaction with β-catenin. Here, we investigated inhibitor of β-catenin and T-cell factor (ICAT), a β-catenin binding protein that inhibits the canonical Wnt/β-catenin signaling pathway, in AR signaling. We demonstrated that expression of ICAT in two AR positive prostate cancer cell lines, LNCaP and LAPC4, augments ligand-dependent AR-mediated transcription. In contrast, short hairpin RNA knockdown of ICAT and β-catenin specifically blocks enhanced AR-mediated transcription by ICAT. Using both stable expression of ICAT and short hairpin RNA knockdown of ICAT expression approaches, we further showed that ICAT enhances expression of endogenous PSA and KLK2, two androgen response genes, and ligand-induced cell growth. In addition, we identified that ICAT and AR can form a ternary complex with β-catenin using in vitro glutathione S-transferase protein pulldown assays. Moreover, we detected the endogenous protein complex containing ICAT, AR, and β-catenin in prostate cancer cells using immunoprecipitation assays. Recruitment of endogenous ICAT onto the promoter region of the human PSA gene, an AR downstream target promoter, was also identified in LNCaP cells. Finally, using in vitro protein binding assays, we examined the effect of full-length and truncated ICAT on the AR-β-catenin interaction and observed that addition of full-length ICAT retained the interaction between β-catenin and AR proteins. Intriguingly, the truncated ICAT comprising the N-terminal helical domain showed a more pronounced effect on β-catenin binding to AR proteins. Our findings suggest a novel molecular mechanism underlying the cross talk between androgen and Wnt signaling pathways.

Structural Differences between the Streptococcus agalactiae Housekeeping and Pilus-Specific Sortases: SrtA and SrtC1

The assembly of pili on the cell wall of Gram-positive bacteria requires transpeptidase enzymes called sortases. In Streptococcus agalactiae, the PI-1 pilus island of strain 2603V/R encodes two pilus-specific sortases (SrtC1 and SrtC2) and three pilins (GBS80, GBS52 and GBS104). Although either pilus-specific sortase is sufficient for the polymerization of the major pilin, GBS80, incorporation of the minor pilins GBS52 and GBS104 into the pilus structure requires SrtC1 and SrtC2, respectively. The S. agalactiae housekeeping sortase, SrtA, whose gene is present at a different location and does not catalyze pilus polymerization, was shown to be involved in cell wall anchoring of pilus polymers. To understand the structural basis of sortases involved in such diverse functions, we determined the crystal structures of S. agalactiae SrtC1 and SrtA. Both enzymes are made of an eight-stranded beta-barrel core with variations in their active site architecture. SrtA exhibits a catalytic triad arrangement similar to that in Streptococcus pyogenes SrtA but different from that in Staphylococcus aureus SrtA. In contrast, the SrtC1 enzyme contains an N-terminal helical domain and a ‘lid’ in its putative active site, which is similar to that seen in Streptococcus pneumoniae pilus-specific sortases, although with subtle differences in positioning and composition. To understand the effect of such differences on substrate recognition, we have also determined the crystal structure of a SrtC1 mutant, in which the conserved DP(W/F/Y) motif was replaced with the sorting signal motif of GBS80, IPNTG. By comparing the structures of WT wild type SrtA and SrtC1 and the ‘lid’ mutant of SrtC1, we propose that structural elements within the active site and the lid may be important for defining the role of specific sortase in pili biogenesis.

Characterizing the N- and C-terminal Small Ubiquitin-like Modifier (SUMO)-interacting Motifs of the Scaffold Protein DAXX

DAXX is a scaffold protein with diverse roles that often depend upon binding SUMO via its N- and/or C-terminal SUMO-interacting motifs (SIM-N and SIM-C). Using NMR spectroscopy, we characterized the in vitro binding properties of peptide models of SIM-N and SIM-C to SUMO-1 and SUMO-2. In each case, binding was mediated by hydrophobic and electrostatic interactions and weakened with increasing ionic strength. Neither isolated SIM showed any significant paralog specificity, and the measured μM range K(D) values of SIM-N toward both SUMO-1 and SUMO-2 were ∼4-fold lower than those of SIM-C. Furthermore, SIM-N bound SUMO-1 predominantly in a parallel orientation, whereas SIM-C interconverted between parallel and antiparallel binding modes on an ms to μs time scale. The differences in affinities and binding modes are attributed to the differences in charged residues that flank the otherwise identical hydrophobic core sequences of the two SIMs. In addition, within its native context, SIM-N bound intramolecularly to the adjacent N-terminal helical bundle domain of DAXX, thus reducing its apparent affinity for SUMO. This behavior suggests a possible autoregulatory mechanism for DAXX. The interaction of a C-terminal fragment of DAXX with an N-terminal fragment of the sumoylated Ets1 transcription factor was mediated by SIM-C. Importantly, this interaction did not involve any direct contacts between DAXX and Ets1, but rather was derived from the non-covalent binding of SIM-C to SUMO-1, which in turn was covalently linked to the unstructured N-terminal segment of Ets1. These results provide insights into the binding mechanisms and hence biological roles of the DAXX SUMO-interacting motifs.

The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

A Nudix enzyme from Bacillus cereus (NCBI RefSeq accession no. NP_831800) catalyzes the hydrolysis of CDP-choline to produce CMP and phosphocholine. Here, we show that in addition, the enzyme has a 3'→5' RNA exonuclease activity. The structure of the free enzyme, determined to a 1.8-Å resolution, shows that the enzyme is an asymmetric dimer. Each monomer consists of two domains, an N-terminal helical domain and a C-terminal Nudix domain. The N-terminal domain is placed relative to the C-terminal domain such as to result in an overall asymmetric arrangement with two distinct catalytic sites: one with an "enclosed" Nudix pyrophosphatase site and the other with a more open, less-defined cavity. Residues that may be important for determining the asymmetry are conserved among a group of uncharacterized Nudix enzymes from Gram-positive bacteria. Our data support a model where CDP-choline hydrolysis is catalyzed by the enclosed Nudix site and RNA exonuclease activity is catalyzed by the open site. CDP-Chase is the first identified member of a novel Nudix family in which structural asymmetry has a profound effect on the recognition of substrates.

CDP-Chase, a CDP-Choline Pyrophosphatase, is a Member of a Novel Nudix Family in Gram-Positive Bacteria

A Nudix enzyme from Bacillus cereus, CDP-Chase, acts as a CDP-choline pyrophosphatase, hydrolyzing the phosphoanhydride bond of CDP-choline to produce CMP and phosphocholine. The structure of the free enzyme, determined to 1.8 A resolution, shows that the enzyme is an asymmetric dimer. Each monomer consists of two domains, an N-terminal helical domain and a C-terminal Nudix domain. The N-terminal domain is placed relative to the C-terminal domain in such a way that produces an overall asymmetry. Residues that may be important for determining the asymmetry are conserved among a group of uncharacterized Nudix enzymes from Gram-positive bacteria. In addition to its Nudix activity, the enzyme has a 3’ to 5’ RNA exonuclease activity. This alternative activity appears to be facilitated by the asymmetry in the protein as the position of the N-terminal domain results in differences in the exposure of the two enzyme active sites. Two single-site mutations, E112A and E163A, were characterized to further investigate the mechanism of the enzyme. E112 is involved in the coordination of catalytic metals in both active sites, and E163 is only in close proximity in one of the active sites. Both mutations abolish CDP-choline pyrophosphatase activity but E112A has a much more profound effect on RNase activity, supporting a model where CDP-choline hydrolysis is catalyzed by one active site of the dimer and RNA exonuclease activity is catalyzed by the other. These data suggest that CDP-Chase is a member of a novel Nudix family in which structural asymmetry has a profound effect on the recognition of substrates by the Nudix enzymatic machinery.

Structural Characterization of the DAXX N-Terminal Helical Bundle Domain and Its Complex with Rassf1C

DAXX is a scaffold protein with diverse roles including transcription and cell cycle regulation. Using NMR spectroscopy, we demonstrate that the C-terminal half of DAXX is intrinsically disordered, whereas a folded domain is present near its N terminus. This domain forms a left-handed four-helix bundle (H1, H2, H4, H5). However, due to a crossover helix (H3), this topology differs from that of the Sin3 PAH domain, which to date has been used as a model for DAXX. The N-terminal residues of the tumor suppressor Rassf1C fold into an amphipathic α helix upon binding this DAXX domain via a shallow cleft along the flexible helices H2 and H5 (K(D) ∼60μM). Based on a proposed DAXX recognition motif as hydrophobic residues preceded by negatively charged groups, we found that peptide models of p53 and Mdm2 also bound the helical bundle. These data provide a structural foundation for understanding the diverse functions of DAXX.

Loop-Sheet Mechanism of Serpin Polymerization Tested by Reactive Center Loop Mutations

The serpin mechanism of protease inhibition involves the rapid and stable incorporation of the reactive center loop (RCL) into central β-sheet A. Serpins therefore require a folding mechanism that bypasses the most stable "loop-inserted" conformation to trap the RCL in an exposed and metastable state. This unusual feature of serpins renders them highly susceptible to point mutations that lead to the accumulation of hyperstable misfolded polymers in the endoplasmic reticulum of secretory cells. The ordered and stable protomer-protomer association in serpin polymers has led to the acceptance of the "loop-sheet" hypothesis of polymerization, where a portion of the RCL of one protomer incorporates in register into sheet A of another. Although this mechanism was proposed 20 years ago, no study has ever been conducted to test its validity. Here, we describe the properties of a variant of α(1)-antitrypsin with a critical hydrophobic section of the RCL substituted with aspartic acid (P8-P6). In contrast to the control, the variant was unable to polymerize when incubated with small peptides or when cleaved in the middle of the RCL (accepted models of loop-sheet polymerization). However, when induced by guanidine HCl or heat, the variant polymerized in a manner indistinguishable from the control. Importantly, the Asp mutations did not affect the ability of the Z or Siiyama α(1)-antitrypsin variants to polymerize in COS-7 cells. These results argue strongly against the loop-sheet hypothesis and suggest that, in serpin polymers, the P8-P6 region is only a small part of an extensive domain swap.

Focus on Molecules: Collagens V and XI

The collagens are a family of 28 extracellular matrix glycoproteins. Collagens are composed of 3 polypeptides, called alpha chains. Each α chain contains at least one stiff, rod-like domain of varying length with a glycine at every third residue that can form a triple helix, i.e., collagenous domain. Collagens V and XI are members of the fibril-forming class of collagens, along with collagens I, II, III, XXIV and XXVII. Collagens V and XI comprise a subclass of regulatory fibrillar collagens that co-assemble with collagens I, II and III. All collagens are trimers composed of 3 α chains with at least one triple helical or collagen (COL) domain. Alpha chains of collagens V (P20908 and P25940) and XI (P12107 and P13942) are encoded by genes with different phylogenetic origins, belonging to different clades or `ancestral groups' (clade 2) from collagens I, II and III (clade 1) or XXIV and XXVII (clade 3). Clade 1 also contains the α2(V) gene (P05997; i.e., the α2 chain of collagen V) and the α1(II) gene, termed α3(XI) when part of a collagen XI trimer (P02458). These genes code for multi-domain polypeptide chains with features common to all fibrillar collagens: a signal peptide; an N-terminal non-collagenous region (NC3); a short N-terminal triple helical domain of interrupted Gly-X–Y repeats (COL2), where X and Y can be any amino acid, but are often proline and hydroxyproline, respectively; a short non-collagenous domain (NC2 or `hinge'); the main triple helical or collagenous domain of continuous (GlyX–Y)n repeats (COL1); and a non-collagenous C-propeptide (NC1) (Fig.1).

Structural mechanism of host Rab1 activation by the bifunctional Legionella type IV effector SidM/DrrA

Bacterial pathogens deliver effector proteins with diverse biochemical activities into host cells, thereby modulating various host functions. Legionella pneumophila hijacks host vesicle trafficking to avoid phagosome-lysosome fusion, a mechanism that is dependent on the Legionella Dot/Icm type IV secretion system. SidM/DrrA, a Legionella type IV effector, is important for the interactions of Legionella-containing vacuoles with host endoplasmic reticulum-derived vesicles. SidM is the only known protein that catalyzes both the exchange of GDP for GTP and GDI displacement from small GTPase Rab1. We determined the crystal structures of SidM alone (residues 317-647) and SidM (residues 193-550) in complex with nucleotide-free WT Rab1. The SidM structure contains an N-terminal helical domain with a potential new function, a Rab1-activation domain, and a C-terminal phosphatidylinositol 4-phosphate-binding P4M domain. The Rab1-activation domain has extensive strong interactions mainly with Rab1 switch I and II regions that undergo substantial conformational changes on SidM binding. Mutations of switch-contacting residues in SidM attenuate both the nucleotide exchange and GDI displacement activities. Structural comparisons of Rab1 in the SidM complex with Rab1-GDP and Ypt1-GDP in the GDI complex identify key conformational changes that disrupt the nucleotide and GDI binding of Rab1. Further biochemical and structural analyses reveal a unique mechanism of coupled GDP release and GDI displacement likely triggered by the SidM-induced drastic displacement of switch I of Rab1.

Geometric Curvature Sensing of Alpha-Helical Proteins

Understanding how specific proteins are recruited to membranes is crucial to understanding key biological functions. Membrane curvature sensing is increasingly recognized as a powerful means for guiding protein localization, coupling protein binding to the local membrane geometry. The mechanisms underlying curvature sensing remain poorly quantified, however. We focus on the two curvature-associated proteins that share an amphipathic alpha-helical structure. The first, SpoVM, is a small (26 residue) protein from Bacillus subtillus that has recently been shown to preferentially bind to convex surfaces [K.S. Ramamurthy et al., Science 323: 1354 (2009)]. The second is the 23 amino acid N-terminal helical domain of Sar1, which initiates the assembly of COPII coated vesicles at the endoplasmic reticulum and which recent work [Parthasarthy Lab, unpublished] has shown to dramatically alter membrane rigidity. Using both microfabricated surfaces that present controlled curvatures to the proteins and optical-trap based assays involving dynamic membrane deformation, we quantify the curvature dependence of the membrane binding affinity of these structurally similar yet functionally disparate proteins.

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers, using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein domains. The N-terminal amphipathic helical domain Ia (residues 1-16) rests on the surface of the lipid membrane with the hydrophobic face of domain Ia embedded in the membrane bilayer interior. The helix comprised of domain Ib (residues 23-30) and transmembrane domain II (residues 31-52) traverses the bilayer with a tilt angle of approximately 24 degrees . The specific interactions between PLN and lipid membranes may represent an additional regulatory element of its inhibitory function. We propose this hybrid method for the simultaneous determination of structure and topology for membrane proteins with compact folds or proteins whose spatial arrangement is dictated by their specific interactions with lipid bilayers.