Biomedical subjects

Johann Deisenhofer

Publications and source records attributed to Johann Deisenhofer.

At least 19 recordsLinked to original sources

PubMed2006

Signal transduction pathway of TonB-dependent transporters.

Transcription of the ferric citrate import system is regulated by ferric citrate binding to the outer membrane transporter FecA. A signal indicating transporter occupancy is relayed across the outer membrane to energy-transducing and regulatory proteins embedded in the cytoplasmic membrane. Because transcriptional activation is not coupled to ferric citrate import, an allosteric mechanism underlies this complex signaling mechanism. Using evolution-based statistical analysis we have identified a sparse but structurally connected network of residues that links distant functional sites in FecA. Functional analyses of these positions confirm their involvement in the mechanism that regulates transcriptional activation in response to ferric citrate binding at the cell surface. This mechanism appears to be conserved and provides the structural basis for the allosteric signaling of TonB-dependent transporters.

Bacterial Proteins↗

PubMed2006

Crystal structure of cryptochrome 3 from Arabidopsis thaliana and its implications for photolyase activity.

Cryptochromes use near-UV/blue light to regulate a variety of growth and adaptive process. Recent biochemical studies demonstrate that the Cryptochrome-Drosophila, Arabidopsis, Synechocystis, Human (Cry-DASH) subfamily of cryptochromes have photolyase activity exclusively for single-stranded cyclobutane pyrimidine dimer (CPD)-containing DNA substrate [Selby C, Sancar A (2006) Proc Natl Acad Sci USA 103:17696-17700]. The crystal structure of cryptochrome 3 from Arabidopsis thaliana (At-Cry3), a member of the Cry-DASH proteins, at 2.1 A resolution, reveals that both the light-harvesting cofactor 5,10-methenyl-tetrahydrofolyl-polyglutamate (MTHF) and the catalytic cofactor flavin adenine dinucleotide (FAD) are noncovalently bound to the protein. The residues responsible for binding of MTHF in At-Cry3 are not conserved in Escherichia coli photolyase but are strongly conserved in the Cry-DASH subfamily of cryptochromes. The distance and orientation between MTHF and flavin adenine dinucleotide in At-Cry3 is similar to that of E. coli photolyase, in conjunction with the presence of electron transfer chain, suggesting the conservation of redox activity in At-Cry3. Two amino acid substitutions and the penetration of three charged side chains into the CPD-binding cavity in At-Cry3 alter the hydrophobic environment that is accommodating the hydrophobic sugar ring and thymine base moieties in class I CPD photolyases. These changes most likely make CPD binding less energetically favorable and, hence, insufficient to compete with pairing and stacking interactions between the CPD and the duplex DNA substrate. Thus, Cry-DASH subfamily proteins may be unable to stabilize CPD flipped out from the duplex DNA substrate but may be able to preserve the DNA repair activity toward single-stranded CPD-containing DNA substrate.

Amino Acid Sequence↗

PubMed2006

Structure of tracheal cytotoxin in complex with a heterodimeric pattern-recognition receptor.

Tracheal cytotoxin (TCT), a naturally occurring fragment of Gram-negative peptidoglycan, is a potent elicitor of innate immune responses in Drosophila. It induces the heterodimerization of its recognition receptors, the peptidoglycan recognition proteins (PGRPs) LCa and LCx, which activates the immune deficiency pathway. The crystal structure at 2.1 angstrom resolution of TCT in complex with the ectodomains of PGRP-LCa and PGRP-LCx shows that TCT is bound to and presented by the LCx ectodomain for recognition by the LCa ectodomain; the latter lacks a canonical peptidoglycan-docking groove conserved in other PGRPs. The interface, revealed in atomic detail, between TCT and the receptor complex highlights the importance of the anhydro-containing disaccharide in bridging the two ectodomains together and the critical role of diaminopimelic acid as the specificity determinant for PGRP interaction.

Amino Acid Sequence↗

PubMed2006

Mechanism of substrate specificity in Bacillus subtilis ResA, a thioredoxin-like protein involved in cytochrome c maturation.

The covalent attachment of heme cofactors to the apo-polypeptides via thioether bonds is unique to the maturation of c-type cytochromes. A number of thiol-disulfide oxidoreductases prepare the apocytochrome for heme insertion in system I and II cytochrome c maturation. Although most thiol-disulfide oxidoreductases are nonspecific, the less common, specific thiol-disulfide oxidoreductases may be key to directing the usage of electrons. Here we demonstrate that unlike other thiol-disulfide oxidoreductases, the protein responsible for reducing oxidized apocytochrome c in Bacillus subtilis, ResA, is specific for cytochrome c550 and utilizes alternate conformations to recognize redox partners. We report solution NMR evidence that ResA undergoes a redox-dependent conformational change between oxidation states, as well as data showing that ResA utilizes a surface cavity present only in the reduced state to recognize a peptide derived from cytochrome c550. Finally, we confirm that ResA is a specific thiol-disulfide oxidoreductase by comparing its reactivity to our mimetic peptide with its reactivity to oxidized glutathione, a nonspecific substrate. This study biochemically demonstrates the specificity of this thiol-disulfide oxidoreductase and enables us to outline a structural mechanism of regulating the usage of electrons in a thiol-disulfide oxidoreductase system.

Bacillus subtilis↗

PubMed2005

Mechanistic insight into the allosteric activation of a ubiquitin-conjugating enzyme by RING-type ubiquitin ligases.

Ubiquitin-conjugating enzymes (E2s) collaborate with the ubiquitin-activating enzyme (E1) and ubiquitin ligases (E3s) to attach ubiquitin to target proteins. RING-containing E3s simultaneously bind to E2s and substrates, bringing them into close proximity and thus facilitating ubiquitination. We show herein that, although the E3-binding site on the human E2 UbcH5b is distant from its active site, two RING-type minimal E3 modules lacking substrate-binding functions greatly stimulate the rate of ubiquitin release from the UbcH5b-ubiquitin thioester. Using statistical coupling analysis and mutagenesis, we identify and characterize clusters of coevolving and functionally linked residues within UbcH5b that span its E3-binding and active sites. Several UbcH5b mutants are defective in their stimulation by E3s despite their abilities to bind to these E3s, to form ubiquitin thioesters, and to release ubiquitin at a basal rate. One such mutation, I37A, is distant from both the active site and the E3-binding site of UbcH5b. Our studies reveal structural determinants for communication between distal functional sites of E2s and suggest that RING-type E3s activate E2s allosterically.

Allosteric Site↗

PubMed2005

NMR structures of the selenoproteins Sep15 and SelM reveal redox activity of a new thioredoxin-like family.

Selenium has significant health benefits, including potent cancer prevention activity and roles in immune function and the male reproductive system. Selenium-containing proteins, which incorporate this essential micronutrient as selenocysteine, are proposed to mediate the positive effects of dietary selenium. Presented here are the solution NMR structures of the selenoprotein SelM and an ortholog of the selenoprotein Sep15. These data reveal that Sep15 and SelM are structural homologs that establish a new thioredoxin-like protein family. The location of the active-site redox motifs within the fold together with the observed localized conformational changes after thiol-disulfide exchange and measured redox potential indicate that they have redox activity. In mammals, Sep15 expression is regulated by dietary selenium, and either decreased or increased expression of this selenoprotein alters redox homeostasis. A physiological role for Sep15 and SelM as thiol-disulfide oxidoreductases and their contribution to the quality control pathways of the endoplasmic reticulum are discussed.

Amino Acid Motifs↗

PubMed2005

Structure of the ectodomain of Drosophila peptidoglycan-recognition protein LCa suggests a molecular mechanism for pattern recognition.

The peptidoglycan-recognition protein LCa (PGRP-LCa) is a transmembrane receptor required for activation of the Drosophila immune deficiency pathway by monomeric Gram-negative peptidoglycan. We have determined the crystal structure of the ectodomain of PGRP-LCa at 2.5-A resolution and found two unique helical insertions in the LCa ectodomain that disrupt an otherwise L-shaped peptidoglycan-docking groove present in all other known PGRP structures. The deficient binding of PGRP-LCa to monomeric peptidoglycan was confirmed by biochemical pull-down assays. Recognition of monomeric peptidoglycan involves both PGRP-LCa and -LCx. We showed that association of the LCa and LCx ectodomains in vitro depends on monomeric peptidoglycan. The presence of a defective peptidoglycan-docking groove, while preserving a unique role in mediating monomeric peptidoglycan induction of immune response, suggests that PGRP-LCa recognizes the exposed structural features of a monomeric muropeptide when the latter is bound to and presented by the ectodomain of PGRP-LCx. Such features include N-acetyl glucosamine and the anhydro bond in the glycan of the muropeptide, which have been demonstrated to be critical for immune stimulatory activity.

Animals↗

PubMed2004

A Drosophila pattern recognition receptor contains a peptidoglycan docking groove and unusual L,D-carboxypeptidase activity.

The Drosophila peptidoglycan recognition protein SA (PGRP-SA) is critically involved in sensing bacterial infection and activating the Toll signaling pathway, which induces the expression of specific antimicrobial peptide genes. We have determined the crystal structure of PGRP-SA to 2.2-A resolution and analyzed its peptidoglycan (PG) recognition and signaling activities. We found an extended surface groove in the structure of PGRP-SA, lined with residues that are highly diverse among different PGRPs. Mutational analysis identified it as a PG docking groove required for Toll signaling and showed that residue Ser158 is essential for both PG binding and Toll activation. Contrary to the general belief that PGRP-SA has lost enzyme function and serves primarily for PG sensing, we found that it possesses an intrinsic L,D-carboxypeptidase activity for diaminopimelic acid-type tetrapeptide PG fragments but not lysine-type PG fragments, and that Ser158 and His42 may participate in the hydrolytic activity. As L,D-configured peptide bonds exist only in prokaryotes, this work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria and provides a possible explanation of how PGRP-SA mediates Toll activation specifically in response to lysine-type PG.

Amino Acid Sequence↗

PubMed2004

Structure of the photolyase-like domain of cryptochrome 1 from Arabidopsis thaliana.

Signals generated by cryptochrome (CRY) blue-light photoreceptors are responsible for a variety of developmental and circadian responses in plants. The CRYs are also identified as circadian blue-light photoreceptors in Drosophila and components of the mammalian circadian clock. These flavoproteins all have an N-terminal domain that is similar to photolyase, and most have an additional C-terminal domain of variable length. We present here the crystal structure of the photolyase-like domain of CRY-1 from Arabidopsis thaliana. The structure reveals a fold that is very similar to photolyase, with a single molecule of FAD noncovalently bound to the protein. The surface features of the protein and the dissimilarity of a surface cavity to that of photolyase account for its lack of DNA-repair activity. Previous in vitro experiments established that the photolyase-like domain of CRY-1 can bind Mg.ATP, and we observe a single molecule of an ATP analog bound in the aforementioned surface cavity, near the bound FAD cofactor. The structure has implications for the signaling mechanism of CRY blue-light photoreceptors.

Adenosine Triphosphate↗

PubMed2004

Metal import through microbial membranes.

Transport systems of Gram-negative bacteria coordinate the passage of metabolites through the outer membrane, periplasm, and the cytoplasmic membrane without compromising the protective properties of the cell envelope. Active transporters orchestrate the import of metals against concentration gradients. These thermodynamically unfavorable processes are coupled to both an electrochemical proton gradient and the hydrolysis of ATP. Crystallographic structures of transport proteins now define in molecular detail most components of an active metal import pathway from Escherichia coli.

Adenosine Triphosphate↗

PubMed2003

Tetramerization and ATP binding by a protein comprising the A, B, and C domains of rat synapsin I.

Synapsins are multidomain proteins that are critical for regulating neurotransmitter release in vertebrates. In the present study, two crystal structures of the C domain of rat synapsin I (rSynI-C) in complex with Ca(2+) and ATP reveal that this protein can form a tetramer and that a flexible loop (the "multifunctional loop") contacts bound ATP. Further experiments were carried out on a protein comprising the A, B, and C domains of rat synapsin I (rSynI-ABC). An ATP-stabilized tetramer of rSynI-ABC is observed during velocity sedimentation and size-exclusion chromatographic experiments. These hydrodynamic results also indicate that the A and B domains exist in an extended conformation. Calorimetric measurements of ATP binding to wild-type and mutant rSynI-ABC demonstrate that the multifunctional loop and a cross-tetramer contact are important for ATP binding. The evidence supports a view of synapsin I as an ATP-utilizing, tetrameric protein made up of monomers that have a flexible, extended N terminus.

Adenosine Triphosphate↗

PubMed2003

Ping-pong cross-validation in real space: a method for increasing the phasing power of a partial model without risk of model bias.

Experimental phases could only be obtained to 4.4 A resolution for crystals of the SecA translocation ATPase. Density modification of these phases exploiting the 65% solvent content of the crystal produced a map from which an approximate backbone model could be built for 80% of the structure. Combining the phases inferred from this partial model with the MIR phases and repeating the density modification produced an improved map from which a more complete backbone model could be built. However, this procedure converged before yielding a map, that allowed unambiguous sequence assignment for the majority of the protein molecule. In order to avoid the likely model bias associated with a speculative attempt at sequence assignment, a real-space cross-validation procedure was employed to facilitate completion of the crystal structure based on partial model phasing. The protein was partitioned into two disjoint sets of residues. Models in which the side chains were built for residues in one of the two sets were used for phase combination and density modification in order to produce improved electron density for interpretation of residues in the other set that had not been included in the model. Residues in the two sets were therefore omitted from the model in alternation except at sites where the side chain could be identified definitively based on phasing with the other set. This ping-pong cross-validation procedure allowed partial model phasing to be used to complete the crystal structure of SecA without being impeded by model bias. These results show that the structure of a large protein molecule can be solved with exclusively low-resolution experimental phase information based on intensive use of partial model phasing and density modification. Real-space cross-validation can be applied to reduce the risk of model bias associated with partial model phasing, streamlining this approach and expanding its range of applicability.

Adenosine Triphosphatases↗

PubMed2003

The low-density lipoprotein receptor: ligands, debates and lore.

Like pieces belonging to a large mosaic, the structures of low-density lipoprotein receptor (LDL-R) modules have been elucidated one by one in recent years. LDL-Rs localized on hepatocytes play an important role in removing cholesterol-transporting LDL particles from the plasma by receptor-mediated endocytosis. Key steps in this process involve the LDL-R binding LDL at neutral pH at the cell surface and, after internalization, releasing it again at acidic pH in the endosomes. How the modules of the LDL-R might interact within the intact receptor to carry out ligand binding and release has been revealed by the recent crystal structure of the extracellular domain of the LDL-R.

Animals↗

PubMed2002

Structure of the LDL receptor extracellular domain at endosomal pH.

The low-density lipoprotein receptor mediates cholesterol homeostasis through endocytosis of lipoproteins. It discharges its ligand in the endosome at pH < 6. In the crystal structure at pH = 5.3, the ligand-binding domain (modules R2 to R7) folds back as an arc over the epidermal growth factor precursor homology domain (the modules A, B, beta propeller, and C). The modules R4 and R5, which are critical for lipoprotein binding, associate with the beta propeller via their calcium-binding loop. We propose a mechanism for lipoprotein release in the endosome whereby the beta propeller functions as an alternate substrate for the ligand-binding domain, binding in a calcium-dependent way and promoting lipoprotein release.

Amino Acid Sequence↗

PubMed2002

Structure of factor-inhibiting hypoxia-inducible factor 1: An asparaginyl hydroxylase involved in the hypoxic response pathway.

Precise regulation of the evolutionarily conserved hypoxia-inducible transcription factor (HIF) ensures proper adaptation to variations in oxygen availability throughout development and into adulthood. Oxygen-dependent regulation of HIF stability and activity are mediated by hydroxylation of conserved proline and asparagine residues, respectively. Because the relevant prolyl and asparginyl hydroxylases use O(2) to effect these posttranslational modifications, these enzymes are implicated as direct oxygen sensors in the mammalian hypoxic response pathway. Here we present the structure of factor-inhibiting HIF-1 (FIH-1), the pertinent asparaginyl hydroxylase involved in hypoxic signaling. Hydroxylation of the C-terminal transactivation domain (CTAD) of HIF by FIH-1 prevents CTAD association with transcriptional coactivators under normoxic conditions. Consistent with other structurally known hydroxylases, FIH-1 is comprised of a beta-strand jellyroll core with both Fe(II) and the cosubstrate 2-oxoglutarate bound in the active site. Details of the molecular contacts at the active site of FIH-1 have been elucidated and provide a platform for future drug design. Furthermore, the structure reveals the presence of a FIH-1 homodimer that forms in solution and is essential for FIH activity.

Amino Acid Sequence↗

PubMed2002

TonB-dependent receptors-structural perspectives.

Plants, bacteria, fungi, and yeast utilize organic iron chelators (siderophores) to establish commensal and pathogenic relationships with hosts and to survive as free-living organisms. In Gram-negative bacteria, transport of siderophores into the periplasm is mediated by TonB-dependent receptors. A complex of three membrane-spanning proteins TonB, ExbB and ExbD couples the chemiosmotic potential of the cytoplasmic membrane with siderophore uptake across the outer membrane. The crystallographic structures of two TonB-dependent receptors (FhuA and FepA) have recently been determined. These outer membrane transporters show a novel fold consisting of two domains. A 22-stranded antiparallel beta-barrel traverses the outer membrane and adjacent beta-strands are connected by extracellular loops and periplasmic turns. Located inside the beta-barrel is the plug domain, composed primarily of a mixed four-stranded beta-sheet and a series of interspersed alpha-helices. Siderophore binding induces distinct local and allosteric transitions that establish the structural basis of signal transduction across the outer membrane and suggest a transport mechanism.

ATP-Binding Cassette Transporters↗

PubMed2002

Nucleotide control of interdomain interactions in the conformational reaction cycle of SecA.

The SecA adenosine triphosphatase (ATPase) mediates extrusion of the amino termini of secreted proteins from the eubacterial cytosol based on cycles of reversible binding to the SecYEG translocon. We have determined the crystal structure of SecA with and without magnesium-adenosine diphosphate bound to the high-affinity ATPase site at 3.0 and 2.7 angstrom resolution, respectively. Candidate sites for preprotein binding are located on a surface containing the SecA epitopes exposed to the periplasm upon binding to SecYEG and are thus positioned to deliver preprotein to SecYEG. Comparisons with structurally related ATPases, including superfamily I and II ATP-dependent helicases, suggest that the interaction geometry of the tandem motor domains in SecA is modulated by nucleotide binding, which is shown by fluorescence anisotropy experiments to reverse an endothermic domain-dissociation reaction hypothesized to gate binding to SecYEG.

Adenosine Diphosphate↗

PubMed2002

Structural basis of gating by the outer membrane transporter FecA.

Siderophore-mediated acquisition systems facilitate iron uptake. We present the crystallographic structure of the integral outer membrane receptor FecA from Escherichia coli with and without ferric citrate at 2.5 and 2.0 angstrom resolution. FecA is composed of three distinct domains: the barrel, plug, and NH2-terminal extension. Binding of ferric citrate triggers a conformational change of the extracellular loops that close the external pocket of FecA. Ligand-induced allosteric transitions are propagated through the outer membrane by the plug domain, signaling the occupancy of the receptor in the periplasm. These data establish the structural basis of gating for receptors dependent on the cytoplasmic membrane protein TonB. By compiling available data for this family of receptors, we propose a mechanism for the energy-dependent transport of siderophores.

Adsorption↗