Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various alpha-keto acids, as well as the cleavage of glycine into CO(2) and NH(3), with concomitant transfer of its alpha-carbon to tetrahydrofolate, generating N(5),N(10)-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochemical studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-l-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA(*)), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming l-methionine in addition to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated derivative of the lipoyl-bearing subunit (E(2)) of the pyruvate dehydrogenase complex [Zhao, S., Miller, J. R., Jian, Y., Marletta, M. A., and Cronan, J. E., Jr. (2003) Chem. Biol. 10, 1293-1302]. Herein, we show that the octanoylated derivative of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, we show that the 5'-dA(*) acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d(15)]H-protein to 5'-deoxyadenosine. Last, our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.

Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-l-methionine (SAM) as the precursor to a high-energy 5'-deoxyadenosyl 5'-radical (5'-dA(*)). In the LS reaction, the 5'-dA(*) is hypothesized to abstract hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM is required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d(15) H-protein of the glycine cleavage system-one of the substrates for LS-has been reported [Cicchillo, R. M., Iwig, D. F., Jones, A. D., Nesbitt, N. M., Baleanu-Gogonea, C., Souder, M. G., Tu, L., and Booker, S. J. (2004) Biochemistry 43, 6378-6386]. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X(3)-C-X(2)-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X(4)-C-X(5)-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 +/- 0.5 irons and 6.4 +/- 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5'-deoxyadenosine (5'-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 +/- 0.1 irons and 3.6 +/- 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 +/- 0.1 irons and 4.7 +/- 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5'-dA or the lipoyl group. Mössbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all associated iron is in the configuration [4Fe-4S](2+). When wild-type LS was reconstituted with (57)Fe and sodium sulfide, it harbored considerably more iron (13.8 +/- 0.6) and sulfide (13.1 +/- 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5'-dA and 0.36 equiv of the lipoyl group. Mössbauer spectroscopy of this protein revealed that only approximately 67% +/- 6% of the iron is in the form of [4Fe-4S](2+) clusters, amounting to 9.2 +/- 0.4 irons and 8.8 +/- 0.1 sulfides or 2 [4Fe-4S](2+) clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the Mössbauer parameters of the clusters associated with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concentration and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-containing motifs.

Spherical high density lipoprotein (sHDL), a key player in reverse cholesterol transport and the most abundant form of HDL, is associated with cardiovascular diseases. Small angle neutron scattering with contrast variation was used to determine the solution structure of protein and lipid components of reconstituted sHDL. Apolipoprotein A1, the major protein of sHDL, forms a hollow structure that cradles a central compact lipid core. Three apoA1 chains are arranged within the low resolution structure of the protein component as one of three possible global architectures: (i) a helical dimer with a hairpin (HdHp), (ii) three hairpins (3Hp), or (iii) an integrated trimer (iT) in which the three apoA1 monomers mutually associate over a portion of the sHDL surface. Cross-linking and mass spectrometry analyses help to discriminate among the three molecular models and are most consistent with the HdHp overall architecture of apoA1 within sHDL.Epidemiological studies firmly establish that circulating levels of high density lipoprotein (HDL) cholesterol and apolipoprotein A1 (apoA1), 2 the major protein constituent of HDL particles, are inversely associated with atherosclerotic heart disease risk (1-3). Moreover, genetic studies further confirm a strong mechanistic link between HDL and apoA1 and cardiovascular disease (4 -8). Defined by its buoyant density characteristics, HDL represent a heterogeneous group of particles with varied lipid composition and protein content that participate in diverse biological functions ranging from lipid transport to innate immune functions. For example, HDL serves as an acceptor of cholesterol from peripheral tissue macrophages and promotes lipid transport through delivery of cholesterol to the liver and steroidogenic tissues (9 -11). HDL also mediates systemic anti-inflammatory and anti-oxidant functions (12-14), and HDL-associated proteins can play critical host defense functions (15). ApoA1 represents nearly three-quarters of the protein content of HDL by mass, and it plays a central functional role in facilitating the numerous biological activities of HDL. Typically present at 2-4 molecules/particle depending upon the degree of HDL maturation, apoA1 serves as the fundamental structural element of the particle (16, 17) and is critical for specific interactions with proteins involved in HDL biogenesis (18, 19), maturation and remodeling (20,21), and recognition by target organ receptors (22,23).Because HDL can be generated in a relatively homogenous form, most structural studies of HDL have focused on reconstituted nascent HDL, a particle composed of two molecules of apoA1 associated with phospholipid and free cholesterol (16,17). Early small angle neutron scattering (SANS) and small angle x-ray scattering (SAXS) studies of nascent HDL particles were reported nearly 3 decades ago and are consistent with an outer protein layer relative to a central lipid core (24,25). Current structural models of nascent HDL have an anti-parallel apoA1 chain orientation and posit that the protein exists...

Abstract In mammalian cells, eight cytoplasmic aminoacyl-tRNA synthetases (AARS), and three non-synthetase proteins, reside in a large multi-tRNA synthetase complex (MSC). AARSs have critical roles in interpretation of the genetic code during protein synthesis, and in non-canonical functions unrelated to translation. Nonetheless, the structure and function of the MSC remain unclear. Partial or complete crystal structures of all MSC constituents have been reported; however, the structure of the holo-MSC has not been resolved. We have taken advantage of cross-linking mass spectrometry (XL-MS) and molecular docking to interrogate the three-dimensional architecture of the MSC in human HEK293T cells. The XL-MS approach uniquely provides structural information on flexibly appended domains, characteristic of nearly all MSC constituents. Using the MS-cleavable cross-linker, disuccinimidyl sulfoxide, inter-protein cross-links spanning all MSC constituents were observed, including cross-links between eight protein pairs not previously known to interact. Intra-protein cross-links defined new structural relationships between domains in several constituents. Unexpectedly, an asymmetric AARS distribution was observed featuring a clustering of tRNA anti-codon binding domains on one MSC face. Possibly, the non-uniform localization improves efficiency of delivery of charged tRNA’s to an interacting ribosome during translation. In summary, we show a highly compact, 3D structural model of the human holo-MSC.

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