Jürgen J. Müller

Adrenodoxin: Structure, stability, and electron transfer properties

Adrenodoxin is an iron‐sulfur protein that belongs to the broad family of the [2Fe‐2S]‐type ferredoxins found in plants, animals and bacteria. Its primary function as a soluble electron carrier between the NADPH‐dependent adrenodoxin reductase and several cytochromes P450 makes it an irreplaceable component of the steroid hormones biosynthesis in the adrenal mitochondria of vertebrates. This review intends to summarize current knowledge about structure, function, and biochemical behavior of this electron transferring protein. We discuss the recently solved first crystal structure of the vertebrate‐type ferredoxin, the truncated adrenodoxin Adx(4‐108), that offers the unique opportunity for better understanding of the structure‐function relationships and stabilization of this protein, as well as of the molecular architecture of [2Fe‐2S] ferredoxins in general. The aim of this review is also to discuss molecular requirements for the formation of the electron transfer complex. Essential comparison between bacterial putidaredoxin and mammalian adrenodoxin will be provided. These proteins have similar tertiary structure, but show remarkable specificity for interactions only with their own cognate cytochrome P450. The discussion will be largely centered on the protein‐protein recognition and kinetics of adrenodoxin dependent reactions. Proteins 2000;40:590–612.

New aspects of electron transfer revealed by the crystal structure of a truncated bovine adrenodoxin, Adx(4–108)

BACKGROUND Adrenodoxin (Adx) is a [2Fe-2S] ferredoxin involved in steroid hormone biosynthesis in the adrenal gland mitochondrial matrix of mammals. Adx is a small soluble protein that transfers electrons from adrenodoxin reductase (AR) to different cytochrome P450 isoforms where they are consumed in hydroxylation reactions. A crystallographic study of Adx is expected to reveal the structural basis for an important electron transfer reaction mediated by a vertebrate [2Fe-2S] ferredoxin. RESULTS The crystal structure of a truncated bovine adrenodoxin, Adx(4-108), was determined at 1.85 A resolution and refined to a crystallographic R value of 0.195. The structure was determined using multiple wavelength anomalous dispersion phasing techniques, making use of the iron atoms in the [2Fe-2S] cluster of the protein. The protein displays the compact (alpha + beta) fold typical for [2Fe-2S] ferredoxins. The polypeptide chain is organized into a large core domain and a smaller interaction domain which comprises 35 residues, including all those previously determined to be involved in binding to AR and cytochrome P450. A small interdomain motion is observed as a structural difference between the two independent molecules in the asymmetric unit of the crystal. Charged residues of Adx(4-108) are clustered to yield a strikingly asymmetric electric potential of the protein molecule. CONCLUSIONS The crystal structure of Adx(4-108) provides the first detailed description of a vertebrate [2Fe-2S] ferredoxin and serves to explain a large body of biochemical studies in terms of a three-dimensional structure. The structure suggests how a change in the redox state of the [2Fe-2S] cluster may be coupled to a domain motion of the protein. It seems likely that the clearly asymmetric charge distribution on the surface of Adx(4-108) and the resulting strong molecular dipole are involved in electrostatic steering of the interactions with AR and cytochrome P450.

Crystal structure of Escherichia coli phage HK620 tailspike: podoviral tailspike endoglycosidase modules are evolutionarily related

Bacteriophage HK620 infects Escherichia coli H and is closely related to Shigella phage Sf6 and Salmonella phage P22. All three Podoviridae recognize and cleave their respective host cell receptor polysaccharide by homotrimeric tailspike proteins. The three proteins exhibit high sequence identity in the 110 residues of their N‐terminal particle‐binding domains, but no apparent sequence similarity in their major, receptor‐binding parts. We have biochemically characterized the receptor‐binding part of HK620 tailspike and determined its crystal structure to 1.38 Å resolution. Its major domain is a right‐handed parallel β‐helix, as in Sf6 and P22 tailspikes. HK620 tailspike has endo‐N‐acetylglucosaminidase activity and produces hexasaccharides of an O18A1‐type O‐antigen. As indicated by the structure of a hexasaccharide complex determined at 1.6 Å resolution, the endoglycosidase‐active sites are located intramolecularly, as in P22, and not between subunits, as in Sf6 tailspike. In contrast, the extreme C‐terminal domain of HK620 tailspike forms a β‐sandwich, as in Sf6 and unlike P22 tailspike. Despite the different folds, structure‐based sequence alignments of the C‐termini reveal motifs conserved between the three proteins. We propose that the tailspike genes of P22, Sf6 and HK620 have a common precursor and are not mosaics of unrelated gene fragments.

An Intersubunit Active Site between Supercoiled Parallel β Helices in the Trimeric Tailspike Endorhamnosidase of Shigella flexneri Phage Sf6

Sf6 belongs to the Podoviridae family of temperate bacteriophages that infect gram-negative bacteria by insertion of their double-stranded DNA. They attach to their hosts specifically via their tailspike proteins. The 1.25 A crystal structure of Shigella phage Sf6 tailspike protein (Sf6 TSP) reveals a conserved architecture with a central, right-handed beta helix. In the trimer of Sf6 TSP, the parallel beta helices form a left-handed, coiled-beta coil with a pitch of 340 A. The C-terminal domain consists of a beta sandwich reminiscent of viral capsid proteins. Further crystallographic and biochemical analyses show a Shigella cell wall O-antigen fragment to bind to an endorhamnosidase active site located between two beta-helix subunits each anchoring one catalytic carboxylate. The functionally and structurally related bacteriophage, P22 TSP, lacks sequence identity with Sf6 TSP and has its active sites on single subunits. Sf6 TSP may serve as an example for the evolution of different host specificities on a similar general architecture.

The Structure of the Trapp Subunit Tpc6 Suggests a Model for a Trapp Subcomplex.

The TRAPP (transport protein particle) complexes are tethering complexes that have an important role at the different steps of vesicle transport. Recently, the crystal structures of the TRAPP subunits SEDL and BET3 have been determined, and we present here the 1.7 Å crystal structure of human TPC6, a third TRAPP subunit. The protein adopts an α/β‐plait topology and forms a dimer. In spite of low sequence similarity, the structure of TPC6 strikingly resembles that of BET3. The similarity is especially prominent at the dimerization interfaces of the proteins. This suggests heterodimerization of TPC6 and BET3, which is shown by in vitro and in vivo association studies. Together with TPC5, another TRAPP subunit, TPC6 and BET3 are supposed to constitute a family of paralogous proteins with closely similar three‐dimensional structures but little sequence similarity among its members.

[53] Scattering studies of ribosomes and ribosomal components

Publisher Summary The chapter discusses the scattering studies of ribosomes and ribosomal components. Scattering methods are suitable to analyze the structural dimensions between those of the electron microscope and X-ray crystal diffraction. The gap in the structural analysis of larger cell organelles (nucleus, mitochondria, plastids), by electron microscopy and of biomacromolecules, (such as proteins or nucleic acids), by X-ray diffraction is closed increasingly by scattering methods such as light scattering (LS), small-angle X-ray scattering (SAXS), wide-angle X-ray scattering (WAXS), and small-angle neutron scattering (SANS). This chapter discusses some theoretical background of the methods, the strategy for computation and interpretation of data, and the progress achieved in the last few years regarding the structure of ribosomal components—RNA and proteins—and ribosomal particles. Most of the studies by far have been performed with ribosomal particles and their constituents from Escherichia coli and only few data are available at present on eukaryotic ribosomes. If the scattering experiment is performed in a way that both the scattering curve of the subunits and that of the associate is measured separately, or if the former is calculated, then information on the distances between the subunits and their relative positions within the associate are obtainable.

Crystal Structure of Klebsiella Sp. Asr1 Phytase Suggests Substrate Binding to a Preformed Active Site that Meets the Requirements of a Plant Rhizosphere Enzyme.

The extracellular phytase of the plant‐associated Klebsiella sp. ASR1 is a member of the histidine‐acid‐phosphatase family and acts primarily as a scavenger of phosphate groups locked in the phytic acid molecule. The Klebsiella enzyme is distinguished from the Escherichia coli phytase AppA by its sequence and phytate degradation pathway. The crystal structure of the phytase from Klebsiella sp. ASR1 has been determined to 1.7 Å resolution using single‐wavelength anomalous‐diffraction phasing. Despite low sequence similarity, the overall structure of Klebsiella phytase bears similarity to other histidine‐acid phosphatases, such as E. coli phytase, glucose‐1‐phosphatase and human prostatic‐acid phosphatase. The polypeptide chain is organized into an α and an α/β domain, and the active site is located in a positively charged cleft between the domains. Three sulfate ions bound to the catalytic pocket of an inactive mutant suggest a unique binding mode for its substrate phytate. Even in the absence of substrate, the Klebsiella phytase is closer in structure to the E. coli phytase AppA in its substrate‐bound form than to phytate‐free AppA. This is taken to suggest a preformed substrate‐binding site in Klebsiella phytase. Differences in habitat and substrate availability thus gave rise to enzymes with different substrate‐binding modes, specificities and kinetics.

Crystal structure of KorA bound to operator DNA: insight into repressor cooperation in RP4 gene regulation

KorA is a global repressor in RP4 which regulates cooperatively the expression of plasmid genes whose products are involved in replication, conjugative transfer and stable inheritance. The structure of KorA bound to an 18-bp DNA duplex that contains the symmetric operator sequence and incorporates 5-bromo-deoxyuridine nucleosides has been determined by multiple-wavelength anomalous diffraction phasing at 1.96-Å resolution. KorA is present as a symmetric dimer and contacts DNA via a helix–turn–helix motif. Each half-site of the symmetric operator DNA binds one copy of the protein in the major groove. As confirmed by mutagenesis, recognition specificity is based on two KorA side chains forming hydrogen bonds to four bases within each operator half-site. KorA has a unique dimerization module shared by the RP4 proteins TrbA and KlcB. We propose that these proteins cooperate with the global RP4 repressor KorB in a similar manner via this dimerization module and thus regulate RP4 inheritance.

The dipole moment of the electron carrier adrenodoxin is not critical for redox partner interaction and electron transfer.

Dipole moments of proteins arise from helical dipoles, hydrogen bond networks and charged groups at the protein surface. High protein dipole moments were suggested to contribute to the electrostatic steering between redox partners in electron transport chains of respiration, photosynthesis and steroid biosynthesis, although so far experimental evidence for this hypothesis was missing. In order to probe this assumption, we changed the dipole moment of the electron transfer protein adrenodoxin and investigated the influence of this on protein-protein interactions and electron transfer. In bovine adrenodoxin, the [2Fe-2S] ferredoxin of the adrenal glands, a dipole moment of 803 Debye was calculated for a full-length adrenodoxin model based on the Adx(4-108) and the wild type adrenodoxin crystal structures. Large distances and asymmetric distribution of the charged residues in the molecule mainly determine the observed high value. In order to analyse the influence of the resulting inhomogeneous electric field on the biological function of this electron carrier the molecular dipole moment was systematically changed. Five recombinant adrenodoxin mutants with successively reduced dipole moment (from 600 to 200 Debye) were analysed for their redox properties, their binding affinities to the redox partner proteins and for their function during electron transfer-dependent steroid hydroxylation. None of the mutants, not even the quadruple mutant K6E/K22Q/K24Q/K98E with a dipole moment reduced by about 70% showed significant changes in the protein function as compared with the unmodified adrenodoxin demonstrating that neither the formation of the transient complex nor the biological activity of the electron transfer chain of the endocrine glands was affected. This is the first experimental evidence that the high dipole moment observed in electron transfer proteins is not involved in electrostatic steering among the proteins in the redox chain.

Comparing crystallographic and solution structures of nitrogenase complexes

A low-resolution structure from X-ray scattering data of Kp1•(ADP•AlF-_4•Kp2)2 predicted a significant change in the iron protein (Kp2) upon complex formation. This has been subsequently confirmed by the crystallographic structure of the complex in the Av system. New scattering results are provided to demonstrate the similarity of this complex in the two species.