A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Comparative structural-functional characterization of recombinant and natural adrenodoxin. Interaction with cytochrome P450scc.

The conditions for heterologous expression of recombinant bovine adrenodoxin in E. coli have been optimized, thus reaching expression levels up to 12-14 micromoles per liter of culture medium. A highly efficient method for purification of this recombinant ferredoxin from the E. coli cells has been developed. The structural-functional properties of the highly purified recombinant protein have been characterized and compared to those of natural adrenodoxin purified from bovine adrenocortical mitochondria. In contrast to natural adrenodoxin, which is characterized by microheterogeneity, the recombinant adrenodoxin is homogeneous as judged by N- and C-terminal amino acid sequencing, and its sequence corresponds to the full-length mature form of adrenodoxin containing 128 amino acid residues. The interactions of the natural and recombinant adrenodoxins with cytochrome P450scc have been studied and compared with respect to: the efficiency of their enzymatic reduction of cytochrome P450scc in a reconstituted system; the ability of the immobilized adrenodoxins to bind cytochrome P450scc; the ability of the adrenodoxins to induce a spectral shift of cytochrome P450scc and to effect the average polarity of exposed tyrosines in the low-spin cytochrome P450scc. The recombinant adrenodoxin is functionally active and in the reduced state as well as at low ionic strength it displays higher affinity to cytochrome P450scc as compared to the natural bovine adrenocortical adrenodoxin. The possible role of the C-terminal sequence of the adrenodoxin molecule in its interaction with cytochrome P450scc as well as the advantages of using the recombinant protein instead of the natural one are discussed.     

Role of Positively Charged Residues Lys267, Lys270, and Arg411 of Cytochrome P450scc (Cyp11A1) in Interaction with Adrenodoxin

Cytochrome P450scc and adrenodoxin are redox proteins of the electron transfer chain of the inner mitochondrial membrane steroid hydroxylases. In the present work site-directed mutagenesis of the charged residues of cytochrome P450scc and adrenodoxin, which might be involved in interaction, was used to study the nature of electrostatic contacts between the hemeprotein and the ferredoxin. The target residues for mutagenesis were selected based on the theoretical model of cytochrome P450scc-adrenodoxin complex and previously reported chemical modification studies of cytochrome P450scc. In the present work, to clarify the molecular mechanism of hemeprotein interaction with ferredoxin, we constructed cytochrome P450scc Lys267, Lys270, and Arg411 mutants and Glu47 mutant of adrenodoxin and analyzed their possible role in electrostatic interaction and the role of these residues in the functional activity of the proteins. Charge neutralization at positions Lys267 or Lys270 of cytochrome P450scc causes no significant effect on the physicochemical and functional properties of cytochrome P450scc. However, cytochrome P450scc mutant Arg411Gln was found to exhibit decreased binding affinity to adrenodoxin and lower activity in the cholesterol side chain cleavage reaction. Studies of the functional properties of Glu47Gln and Glu47Arg adrenodoxin mutants indicate that a negatively charged residue in the loop covering the Fe2S2 cluster, being important for maintenance of the correct architecture of these structural elements of ferredoxin, is not directly involved in electrostatic interaction with cytochrome P450scc. Moreover, our results indicate the presence of at least two different binding (contact) sites on the proximal surface of cytochrome P450scc with different electrostatic input to interaction with adrenodoxin. In the binary complex, the positively charged sites of the proximal surface of cytochrome P450scc well correspond to the two negatively charged sites of adrenodoxin: the "interaction" domain site and the "core" domain site.     

Site-Directed Mutagenesis of Cytochrome P450scc. II. Effect of Replacement of the Arg425 and Arg426 Residues on the Structural and Functional Properties of the Cytochrome P450scc

Cytochrome P450-dependent monooxygenases, in spite of their wide distribution, can be simply divided into a few groups differing in the location of the electron transfer chain and their composition. The two main groups of cytochrome P450-dependent monooxygenases are the mitochondrial and microsomal enzymes. While in two-component microsomal cytochrome P450-dependent monooxygenases electrons are supplied to cytochrome P450 by a flavoprotein (NADPH-cytochrome P450 reductase), in three-component mitochondrial monooxygenases the electrons are supplied to cytochrome P450 by a low molecular weight protein (ferredoxin). The interaction of cytochrome P450 with NADPH-cytochrome P450 reductase and ferredoxin is the subject of intensive studies. Using chemical modification, chemical cross-linking, and sitedirected mutagenesis, we identified surface exposed positively charged residues of cytochrome P450scc which might be important for interaction with adrenodoxin. Theoretical analysis of the distribution of surface electrostatic potential in cytochrome P450 indicates that in contrast to microsomal monooxygenases, cytochromes P450 of mitochondrial type, and cholesterol side-chain cleavage cytochrome P450 (P450scc) in part, carry on the proximal surface an evidently positively charged site that is formed by residues Arg425 and Arg426. In the present work, to estimate the functional role of Arg425 and Arg426 of cytochrome P450scc, we used site-directed mutagenesis to replace these residues with glutamine. The results indicate that residues Arg425 and Arg426 are involved in the formation of a heme-binding center and electrostatic interaction of cytochrome P450scc with its physiological electron-transfer partner, adrenodoxin.     

Mechanism of steroidogenic electron transport: Role of conserved Glu429 in destabilization of CYP11A1-Adrenodoxin complex

In the present work the role of conserved residue E429 of cytochrome P45011A1 has been studied. The charge neutralization of E429Q results in 3-fold decrease of K _d as well as V _max compared to the wild type hemoprotein indicating tighter binding and, as the result, the impaired dissociation of oxidized adrenodoxin from the complex. As cytochrome P45011A1-adrenodoxin complex formation is driven primarily by electrostatic interactions, the low activity of E429Q mutant is completely restored to that of wild type hemoprotein by increasing of ionic strength. The charge neutralization of the corresponding residue of rat cytochrome P45011B2 has the same effect: the activity is 10-fold decreased but it is restored by increasing of ionic strength without effect on the ratio of products formed. Thus, this is the first report on identification of residues involved in modulation of dissociation of redox partner from the complex with cytochrome P450s.     

Structural analysis of CYP2R1 in complex with vitamin D3

The activation of vitamin D to its hormonal form is mediated by cytochrome P450 enzymes. CYP2R1 catalyzes the initial step converting vitamin D into 25-hydroxyvitamin D. A CYP2R1 gene mutation causes an inherited form of rickets due to 25-hydroxylase deficiency. To understand the narrow substrate specificity of CYP2R1 we obtained the hemeprotein in a highly purified state, confirmed the enzyme as a vitamin D 25-hydroxylase, and solved the crystal structure of CYP2R1 in complex with vitamin D₃. The CYP2R1 structure adopts a closed conformation with the substrate access channel being covered by the ordered B′-helix and slightly opened to the surface, which defines the substrate entrance point. The active site is lined by conserved, mostly hydrophobic residues. Vitamin D₃ is bound in an elongated conformation with the aliphatic side-chain pointing toward the heme. The structure reveals the secosteroid binding mode in an extended active site and allows rationalization of the molecular basis of the inherited rickets associated with CYP2R1.     

Molecular identification of adrenal inner zone antigen as a heme-binding protein

The adrenal inner zone antigen (IZA), which reacts specifically with a monoclonal antibody raised against the fasciculata and reticularis zones of the rat adrenal, was previously found to be identical with a protein variously named 25-Dx and membrane-associated progesterone receptor. IZA was purified as a glutathione S-transferase-fused or His(6)-fused protein, and its molecular properties were studied. The UV-visible absorption and EPR spectra of the purified protein showed that IZA bound a heme chromophore in high-spin type. Analysis of the heme indicated that it is of the b type. Site-directed mutagenesis studies were performed to identify the amino-acid residues that bind the heme to the protein. The results suggest that two Tyr residues, Tyr107 and Tyr113, and a peptide stretch, D99-K102, were important for anchoring the heme into a hydrophobic pocket. The effect of IZA on the steroid 21-hydroxylation reaction was investigated in COS-7 cell expression systems. The results suggest that the coexistence of IZA with CYP21 enhances 21-hydroxylase activity.     

Structural characterization of human cholesterol 7α-hydroxylase

Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B′ helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.