Bivalent recognition of fatty acyl-CoA by a human integral membrane palmitoyltransferase.

S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein. Protein S-acylation intersects with almost all areas of human physiology, and several DHHC enzymes are considered as possible therapeutic targets against diseases such as cancer. These efforts would greatly benefit from a detailed understanding of the molecular basis for this crucial enzymatic reaction. Here, we combine X-ray crystallography with all-atom molecular dynamics simulations to elucidate the structure of the precatalytic complex of human DHHC20 in complex with palmitoyl CoA. The resulting structure reveals that the fatty acyl chain inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, whereas the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions. Biochemical experiments corroborate the predictions from our structural model. We show, using both computational and experimental analyses, that palmitoyl CoA acts as a bivalent ligand where the interaction of the DHHC enzyme with both the fatty acyl chain and the CoA headgroup is important for catalytic chemistry to proceed. This bivalency explains how, in the presence of high concentrations of free CoA under physiological conditions, DHHC enzymes can efficiently use palmitoyl CoA as a substrate for autoacylation.

In vitro reconstitution of Wnt acylation reveals structural determinants of substrate recognition by the acyltransferase human Porcupine.

Wnt proteins regulate a large number of processes, including cellular growth, differentiation, and tissue homeostasis, through the highly conserved Wnt signaling pathway in metazoans. Porcupine (PORCN) is an endoplasmic reticulum (ER)-resident integral membrane enzyme that catalyzes posttranslational modification of Wnts with palmitoleic acid, an unsaturated lipid. This unique form of lipidation with palmitoleic acid is a vital step in the biogenesis and secretion of Wnt, and PORCN inhibitors are currently in clinical trials for cancer treatment. However, PORCN-mediated Wnt lipidation has not been reconstituted in vitro with purified enzyme. Here, we report the first successful purification of human PORCN and confirm, through in vitro reconstitution with the purified enzyme, that PORCN is necessary and sufficient for Wnt acylation. By systematically examining a series of substrate variants, we show that PORCN intimately recognizes the local structure of Wnt around the site of acylation. Our in vitro assay enabled us to examine the activity of PORCN with a range of fatty acyl-CoAs with varying length and unsaturation. The selectivity of human PORCN across a spectrum of fatty acyl-CoAs suggested that the kink in the unsaturated acyl chain is a key determinant of PORCN-mediated catalysis. Finally, we show that two putative PORCN inhibitors that were discovered with cell-based assays indeed target human PORCN. Together, these results provide discrete, high-resolution biochemical insights into the mechanism of PORCN-mediated Wnt acylation and pave the way for further detailed biochemical and structural studies.

Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution.

GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.

The molecular mechanism of DHHC protein acyltransferases.

Protein S-acylation is a reversible lipidic posttranslational modification where a fatty acid chain is covalently linked to cysteine residues by a thioester linkage. A family of integral membrane enzymes known as DHHC protein acyltransferases (DHHC-PATs) catalyze this reaction. With the rapid development of the techniques used for identifying lipidated proteins, the repertoire of S-acylated proteins continues to increase. This, in turn, highlights the important roles that S-acylation plays in human physiology and disease. Recently, the first molecular structures of DHHC-PATs were determined using X-ray crystallography. This review will comment on the insights gained on the molecular mechanism of S-acylation from these structures in combination with a wealth of biochemical data generated by researchers in the field.

An Improved Strategy for Fluorescent Tagging of Membrane Proteins for Overexpression and Purification in Mammalian Cells.

An essential prerequisite for in vitro biochemical or structural studies is a construct that is amenable to high level expression and purification and is biochemically "well-behaved". In the field of membrane protein research, the use of green fluorescent protein (GFP) to monitor and optimize the heterologous expression in different hosts has radically changed the ease of streamlining and multiplexing the testing of a large number of candidate constructs. This is achieved by genetically fusing the fluorescent proteins to the N- or C-terminus of the proteins of interest to act as reporters which can then be followed by methods such as microscopy, spectroscopy, or in-gel fluorescence. Nonetheless, a systematic study on the effect of GFP and its spectral variants on the expression and yields of recombinant membrane proteins is lacking. In this study, we genetically appended four common fluorescent protein tags, namely, mEGFP, mVenus, mCerulean, and mCherry, to the N- or C-terminus of different membrane proteins and assessed their expression in mammalian cells by fluorescence-detection size exclusion chromatography (FSEC) and protein purification. We find that, of the four fluorescent proteins, tagging with mVenus systematically results in higher expression levels that translates to higher yields in preparative purifications, thus making a case for switching to this yellow spectral variant as a better fusion tag.

CCL22 mutations drive natural killer cell lymphoproliferative disease by deregulating microenvironmental crosstalk.

Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is characterized by clonal expansion of natural killer (NK) cells where the underlying genetic mechanisms are incompletely understood. In the present study, we report somatic mutations in the chemokine gene CCL22 as the hallmark of a distinct subset of CLPD-NK. CCL22 mutations were enriched at highly conserved residues, mutually exclusive of STAT3 mutations and associated with gene expression programs that resembled normal CD16dim/CD56bright NK cells. Mechanistically, the mutations resulted in ligand-biased chemokine receptor signaling, with decreased internalization of the G-protein-coupled receptor (GPCR) for CCL22, CCR4, via impaired ß-arrestin recruitment. This resulted in increased cell chemotaxis in vitro, bidirectional crosstalk with the hematopoietic microenvironment and enhanced NK cell proliferation in vivo in transgenic human IL-15 mice. Somatic CCL22 mutations illustrate a unique mechanism of tumor formation in which gain-of-function chemokine mutations promote tumorigenesis by biased GPCR signaling and dysregulation of microenvironmental crosstalk.

Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15.

The extracellular signal-regulated protein kinase, ERK2, fully activated by phosphorylation and without a His(6) tag, shows little tendency to dimerize with or without either calcium or magnesium ions when analyzed by light scattering or analytical ultracentrifugation. Light scattering shows that ~90% of ERK2 is monomeric. Sedimentation equilibrium data (obtained at 4.8-11.2 µM ERK2) with or without magnesium (10 mM) are well described by an ideal one-component model with a fitted molar mass of 40180 ± 240 Da (without Mg(2+) ions) or 41290 ± 330 Da (with Mg(2+) ions). These values, close to the sequence-derived mass of 41711 Da, indicate that no significant dimerization of ERK2 occurs in solution. Analysis of sedimentation velocity data for a 15 µM solution of ERK2 with an enhanced van Holde-Weischet method determined the sedimentation coefficient (s) to be ~3.22 S for activated ERK2 with or without 10 mM MgCl(2). The frictional coefficient ratio (f/f(0)) of 1.28 calculated from the sedimentation velocity and equilibrium data is close to that expected for an ~42 kDa globular protein. The translational diffusion coefficient of ~8.3 × 10(-7) cm(2) s(-1) calculated from the experimentally determined molar mass and sedimentation coefficient agrees with the value determined by dynamic light scattering in the absence and presence of calcium or magnesium ions and a value determined by NMR spectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins [Casar, B., et al. (2008) Mol. Cell 31, 708-721]. Our light scattering data show, however, that ERK2 forms a strong 1:1 complex of ~57 kDa with the cytoplasmic scaffold protein PEA-15. Thus, ERK2 binds PEA-15 as a monomer. Our data provide strong evidence that ERK2 is monomeric under physiological conditions. Analysis of the same ERK2 construct with the nonphysiological His(6) tag shows substantial dimerization under the same ionic conditions.

Indefinite noncooperative self-association of chicken deoxy hemoglobin D.

The minor tetrameric hemoglobin (Hb), Hb D, of chicken red blood cells self-associates upon deoxygenation. This self-association enhances the cooperativity of oxygen binding. The maximal Hill coefficient is greater than 4 at high Hb concentrations. Previous measurements at low Hb concentrations were consistent with a monomer-to-dimer equilibrium and an association constant of ∼1.3-1.6 × 10(4) M(-1). Here, the Hb tetramer is considered as the monomer. However, new results indicate that the association extends beyond the dimer. We show by combination of Hb oligomer modeling and sedimentation velocity analyses that the data can be well described by an indefinite noncooperative or isodesmic association model. In this model, the deoxy Hb D associates noncooperatively to give a linear oligomeric chain with an equilibrium association constant of 1.42 × 10(4) M(-1) at 20°C for each step. The data are also well described by a monomer-dimer-tetramer equilibrium model with monomer-to-dimer and dimer-to-tetramer association constants of 1.87 and 1.03 × 10(4) M(-1) at 20°C, respectively. A hybrid recombinant Hb D was prepared with recombinant α(D)-globin and native ß-globin to give a Hb D tetramer (α(2)(D)ß(2)). This rHb D undergoes decreased deoxygenation-dependent self-association compared with the native Hb D. Residue glutamate 138 has previously been proposed to influence intertetramer interactions. Our results with recombinant Hb D show that Glu138 plays no role in deoxy Hb D intertetramer interactions.

Structure of Human ATG9A, the Only Transmembrane Protein of the Core Autophagy Machinery.

Autophagy is a catabolic process involving capture of cytoplasmic materials into double-membraned autophagosomes that subsequently fuse with lysosomes for degradation of the materials by lysosomal hydrolases. One of the least understood components of the autophagy machinery is the transmembrane protein ATG9. Here, we report a cryoelectron microscopy structure of the human ATG9A isoform at 2.9-Å resolution. The structure reveals a fold with a homotrimeric domain-swapped architecture, multiple membrane spans, and a network of branched cavities, consistent with ATG9A being a membrane transporter. Mutational analyses support a role for the cavities in the function of ATG9A. In addition, structure-guided molecular simulations predict that ATG9A causes membrane bending, explaining the localization of this protein to small vesicles and highly curved edges of growing autophagosomes.

Component D of chicken hemoglobin and the hemoglobin of the embryonic Tammar wallaby (Macropus eugenii) self-associate upon deoxygenation: Effect on oxygen binding.

Extensive measurements of oxygen binding by some vertebrate hemoglobins (Hbs) have suggested an unusually high degree of cooperativity with reported Hill coefficients, n(H), greater than 4.0. We have reexamined this possibility of "super-cooperativity" with chicken Hb components A (alpha(A) (2)beta(2)) and D (alpha(D) (2)beta(2)). Prior studies have shown that component D but not A self-associates to dimers of tetramers upon deoxygenation. This self-association is reflected in the oxygen equilibrium of Hb D which shows a maximal n(H), greater than 4.0 at approximately 4 mM heme concentration. In contrast, component A has maximal n(H) value below 3. The value of the maximal n(H) for Hb D increases linearly with the fraction of octamer present in the deoxy Hb. We anticipate that deoxygenation-dependent self-association will be shown to be a general property of Hb D from birds and reptiles. Neither oxygen equilibria nor sedimentation measurements show any evidence that components A and D interact to form a complex when deoxygenated. We have also reexamined the oxygen equilibria of Hbs of an embryonic marsupial, the wallaby. The equilibria in red cells have been reported to have Hill coefficients as high as 5-6. Although our oxygen equilibrium measurements of solutions of unfractionated wallaby Hb at a concentration of approximately 1 mM show no n(H) values greater than approximately 3.0, sedimentation velocity measurements provide clear evidence for deoxygenation-dependent self-association.

Fatty acyl recognition and transfer by an integral membrane S-acyltransferase.

DHHC (Asp-His-His-Cys) palmitoyltransferases are eukaryotic integral membrane enzymes that catalyze protein palmitoylation, which is important in a range of physiological processes, including small guanosine triphosphatase (GTPase) signaling, cell adhesion, and neuronal receptor scaffolding. We present crystal structures of two DHHC palmitoyltransferases and a covalent intermediate mimic. The active site resides at the membrane-cytosol interface, which allows the enzyme to catalyze thioester-exchange chemistry by using fatty acyl-coenzyme A and explains why membrane-proximal cysteines are candidates for palmitoylation. The acyl chain binds in a cavity formed by the transmembrane domain. We propose a mechanism for acyl chain-length selectivity in DHHC enzymes on the basis of cavity mutants with preferences for shorter and longer acyl chains.