Biomedical subjects

Chad A Brautigam

Publications and source records attributed to Chad A Brautigam.

11 recordsLinked to original sources

PubMed2007

Structural analysis of Xanthomonas XopD provides insights into substrate specificity of ubiquitin-like protein proteases.

XopD (Xanthomonas outer protein D), a type III secreted effector from Xanthomonas campestris pv. vesicatoria, is a desumoylating enzyme with strict specificity for its plant small ubiquitin-like modifier (SUMO) substrates. Based on SUMO sequence alignments and peptidase assays with various plant, yeast, and mammalian SUMOs, we identified residues in SUMO that contribute to XopD/SUMO recognition. Further predictions regarding the enzyme/substrate specificity were made by solving the XopD crystal structure. By incorporating structural information with sequence alignments and enzyme assays, we were able to elucidate determinants of the rigid SUMO specificity exhibited by the Xanthomonas virulence factor XopD.

Amino Acid Sequence↗

PubMed2006

Crystal structure of the Tp34 (TP0971) lipoprotein of treponema pallidum: implications of its metal-bound state and affinity for human lactoferrin.

The Tp34 (TP0971) membrane lipoprotein of Treponema pallidum, an obligate human pathogen and the agent of syphilis, was previously reported to have lactoferrin binding properties. Given the non-cultivatable nature of T. pallidum, a structure-to-function approach was pursued to clarify further potential relationships between the Tp34 structural and biochemical properties and its propensity to bind human lactoferrin. The crystal structure of a nonacylated, recombinant form of Tp34 (rTp34), solved to a resolution of 1.9A(,) revealed two metaloccupied binding sites within a dimer; the identity of the ion most likely was zinc. Residues from both of the monomers contributed to the interfacial metal-binding sites; a novel feature was that the delta-sulfur of methionine coordinated the zinc ion. Analytical ultracentrifugation showed that, in solution, rTp34 formed a metal-stabilized dimer and that rTp34 bound human lactoferrin with a stoichiometry of 2:1. Isothermal titration calorimetry further revealed that rTp34 bound human lactoferrin at high (submicromolar) affinity. Finally, membrane topology studies revealed that native Tp34 is not located on the outer surface (outer membrane) of T. pallidum but, rather, is periplasmic. How propensity of Tp34 to bind zinc and the iron-sequestering lactoferrin may relate overall to the biology of T. pallidum infection in humans is discussed.

Crystallography, X-Ray↗

PubMed2006

A synchronized substrate-gating mechanism revealed by cubic-core structure of the bovine branched-chain alpha-ketoacid dehydrogenase complex.

The dihydrolipoamide acyltransferase (E2b) component of the branched-chain alpha-ketoacid dehydrogenase complex forms a cubic scaffold that catalyzes acyltransfer from S-acyldihydrolipoamide to CoA to produce acyl-CoA. We have determined the first crystal structures of a mammalian (bovine) E2b core domain with and without a bound CoA or acyl-CoA. These structures reveal both hydrophobic and the previously unreported ionic interactions between two-fold-related trimers that build up the cubic core. The entrance of the dihydrolipoamide-binding site in a 30-A long active-site channel is closed in the apo and acyl-CoA-bound structures. CoA binding to one entrance of the channel promotes a conformational change in the channel, resulting in the opening of the opposite dihydrolipoamide gate. Binding experiments show that the affinity of the E2b core for dihydrolipoamide is markedly increased in the presence of CoA. The result buttresses the model that CoA binding is responsible for the opening of the dihydrolipoamide gate. We suggest that this gating mechanism synchronizes the binding of the two substrates to the active-site channel, which serves as a feed-forward switch to coordinate the E2b-catalyzed acyltransfer reaction.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2006

Structural insight into interactions between dihydrolipoamide dehydrogenase (E3) and E3 binding protein of human pyruvate dehydrogenase complex.

The 9.5 MDa human pyruvate dehydrogenase complex (PDC) utilizes the specific dihydrolipoamide dehydrogenase (E3) binding protein (E3BP) to tether the essential E3 component to the 60-meric core of the complex. Here, we report crystal structures of the binding domain (E3BD) of human E3BP alone and in complex with human E3 at 1.6 angstroms and 2.2 angstroms, respectively. The latter structure shows that residues from E3BD contact E3 across its 2-fold axis, resulting in one E3BD binding site on the E3 homodimer. Negligible conformational changes occur in E3BD upon its high-affinity binding to E3. Modifications of E3BD residues at the center of the E3BD/E3 interface impede E3 binding far more severely than those of residues on the periphery, validating the "hot spot" paradigm for protein interactions. A cluster of disease-causing E3 mutations located near the center of the E3BD/E3 interface prevents the efficient recruitment of these E3 variants by E3BP into the PDC, leading to the dysfunction of the PDC catalytic machine.

Amino Acid Sequence↗

PubMed2006

The PnrA (Tp0319; TmpC) lipoprotein represents a new family of bacterial purine nucleoside receptor encoded within an ATP-binding cassette (ABC)-like operon in Treponema pallidum.

Treponema pallidum, the bacterial agent of syphilis, cannot be cultivated in vitro. This constraint has severely impeded the study of the membrane biology of this complex human pathogen. A structure-to-function approach thus was adopted as a means of discerning the likely function of Tp0319, a 35-kDa cytoplasmic membrane-associated lipoprotein of T. pallidum formerly designated as TmpC. A 1.7-A crystal structure showed that recombinant Tp0319 (rTp0319) consists of two alpha/beta domains, linked by three crossovers, with a deep cleft between them akin to ATP-binding cassette (ABC) receptors. In the cleft, a molecule of inosine was bound. Isothermal titration calorimetry demonstrated that rTp0319 specifically binds purine nucleosides (dissociation constant (Kd) approximately 10(-7) M). This predilection for purine nucleosides by rTp0319 is consistent with its likely role as a receptor component of a cytoplasmic membrane-associated transporter system. Reverse transcription-PCR analysis of RNA isolated from rabbit tissue-extracted T. pallidum additionally showed that tp0319 is transcriptionally linked to four other downstream open reading frames, thereby supporting the existence of an ABC-like operon (tp0319-0323). We herein thus re-name tp0319 as purine nucleoside receptor A (pnrA), with its operonic partners tp0320-0323 designated as pnrB-E, respectively. Our study not only infers that PnrA transports purine nucleosides essential for the survival of T. pallidum within its obligate human host, but to our knowledge, this is the first description of an ABC-type nucleoside transport system in any bacterium. PnrA has been grouped with a functionally uncharacterized protein family (HBG016869), thereby implying that other members of the family may have similar nucleoside-binding function(s).

Adenosine Triphosphate↗

PubMed2006

A versatile conformational switch regulates reactivity in human branched-chain alpha-ketoacid dehydrogenase.

The dehydrogenase/decarboxylase (E1b) component of the 4 MD human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is a thiamin diphosphate (ThDP)-dependent enzyme. We have determined the crystal structures of E1b with ThDP bound intermediates after decarboxylation of alpha-ketoacids. We show that a key tyrosine residue in the E1b active site functions as a conformational switch to reduce the reactivity of the ThDP cofactor through interactions with its thiazolium ring. The intermediates do not assume the often-postulated enamine state, but likely a carbanion state. The carbanion presumably facilitates the second E1b-catalyzed reaction, involving the transfer of an acyl moiety from the intermediate to a lipoic acid prosthetic group in the transacylase (E2b) component of the BCKDC. The tyrosine switch further remodels an E1b loop region to promote E1b binding to E2b. Our results illustrate the versatility of the tyrosine switch in coordinating the catalytic events in E1b by modulating the reactivity of reaction intermediates.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2005

Crystal structure of human dihydrolipoamide dehydrogenase: NAD+/NADH binding and the structural basis of disease-causing mutations.

Human dihydrolipoamide dehydrogenase (hE3) is an enzymatic component common to the mitochondrial alpha-ketoacid dehydrogenase and glycine decarboxylase complexes. Mutations to this homodimeric flavoprotein cause the often-fatal human disease known as E3 deficiency. To catalyze the oxidation of dihydrolipoamide, hE3 uses two molecules: non-covalently bound FAD and a transiently bound substrate, NAD+. To address the catalytic mechanism of hE3 and the structural basis for E3 deficiency, the crystal structures of hE3 in the presence of NAD+ or NADH have been determined at resolutions of 2.5A and 2.1A, respectively. Although the overall fold of the enzyme is similar to that of yeast E3, these two structures differ at two loops that protrude from the proteins and at their FAD-binding sites. The structure of oxidized hE3 with NAD+ bound demonstrates that the nicotinamide moiety is not proximal to the FAD. When NADH is present, however, the nicotinamide base stacks directly on the isoalloxazine ring system of the FAD. This is the first time that this mechanistically requisite conformation of NAD+ or NADH has been observed in E3 from any species. Because E3 structures were previously available only from unicellular organisms, speculations regarding the molecular mechanisms of E3 deficiency were based on homology models. The current hE3 structures show directly that the disease-causing mutations occur at three locations in the human enzyme: the dimer interface, the active site, and the FAD and NAD(+)-binding sites. The mechanisms by which these mutations impede the function of hE3 are discussed.

Binding Sites↗

PubMed2004

Side chain and backbone contributions of Phe508 to CFTR folding.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

Animals↗

PubMed2004

Structural evidence that the 32-kilodalton lipoprotein (Tp32) of Treponema pallidum is an L-methionine-binding protein.

A structure-to-function approach was undertaken to gain insights into the potential function of the 32-kDa membrane lipoprotein (Tp32) of Treponema pallidum, the syphilis bacterium. The crystal structure of rTp32 (determined at a resolution of 1.85 A) shows that the organization of rTp32 is similar to other periplasmic ligand-binding proteins (PLBPs), in that it consists of two alpha/beta domains, linked by two crossovers, with a binding pocket between them. In the pocket, a molecule of L-methionine was detected in the electron density map. Residues from both domains interact with the ligand. One of the crossover regions is comprised of a 3(10)-helix, a feature not typical in other ligand-binding proteins. Sequence comparison shows strong similarity to other hypothetical methionine-binding proteins. Together, the data support the notion that rTp32 is a component of a periplasmic methionine uptake transporter system in T. pallidum.

Apolipoproteins↗

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↗

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↗