Application of a genetic algorithm in the conformational analysis of methylene-acetal-linked thymine dimers in DNA: Comparison with distance geometry calculations

The three-dimensional spatial structure of a methylene-acetal-linked thymine dimer presentin a 10 base-pair (bp) sense–antisense DNA duplex was studied with a geneticalgorithm designed to interpret NOE distance restraints. Trial solutions were represented bytorsion angles. This means that bond angles for the dimer trial structures are kept fixed duringthe genetic algorithm optimization. Bond angle values were extracted from a 10 bpsense–antisense duplex model that was subjected to energy minimization by means ofa modified AMBER force field. A set of 63 proton–proton distance restraints definingthe methylene-acetal-linked thymine dimer was available. The genetic algorithm minimizesthe difference between distances in the trial structures and distance restraints. A largeconformational search space could be covered in the genetic algorithm optimization byallowing a wide range of torsion angles. The genetic algorithm optimization in all cases ledto one family of structures. This family of the methylene-acetal-linked thymine dimer in theduplex differs from the family that was suggested from distance geometry calculations. It isdemonstrated that the bond angle geometry around the methylene-acetal linkage plays animportant role in the optimization.     

DDB2 promotes chromatin decondensation at UV-induced DNA damage

Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A-RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)-dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]-ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains.     

Transgenic mice expressing dominant-negative activin receptor IB in forebrain neurons reveal novel functions of activin at glutamatergic synapses

The transforming growth factor beta family member activin is an important regulator of development and tissue repair. It is strongly up-regulated after acute injury to the adult brain, and application of exogenous activin protects neurons in several lesion models. To explore the role of endogenous activin in the normal and acutely damaged brain, we generated transgenic mice expressing a dominant-negative activin receptor IB (dnActRIB) mutant in forebrain neurons. The functionality of the transgene was verified in vivo. Hippocampal neurons from dnActRIB mice were significantly more vulnerable to intracerebroventricular injection of the excitotoxin kainic acid than those from control littermates, indicating a crucial role of endogenous activin in the rescue of neurons from excitotoxic insult. Because dnActRIB is only expressed in neurons, but not in glial cells, activin affords protection at least in part through a direct action on endangered neurons. Unexpectedly, the transgenic mice also revealed a prominent novel role of activin in glutamatergic neurotransmission in the intact adult brain. Electrophysiologic examination of excitatory synapses onto CA1 pyramidal cells in hippocampal slices of dnActRIB mice showed a reduced NMDA current response, which was associated with impaired long term potentiation. This is the first demonstration that activin receptor signaling is essential to optimize the performance of neuronal circuits in the mature brain under physiological conditions.     

Roles of His291-alpha and His146-beta' in the reductive acylation reaction catalyzed by human branched-chain alpha-ketoacid dehydrogenase: refined phosphorylation loop structure in the active site

We report here that alterations of either His291-alpha or His146-beta' in the active site of human branched-chain alpha-ketoacid dehydrogenase (E1b) impede both the decarboxylation and the reductive acylation reactions catalyzed by E1b as well as the binding of cofactor thiamin diphosphate (ThDP). In a refined human E1b active-site structure, His291-alpha, which aligns with His407 in Escherichia coli pyruvate dehydrogenase and His263 in yeast transketolase, is on a largely ordered phosphorylation loop. The imidazole ring of His291-alpha in E1b coordinates to the terminal phosphate oxygen atoms of bound ThDP. The N3 atom of wild-type His146-beta', which can be protonated, binds a water molecule and points toward the aminopyrimidine ring of ThDP. Remarkably, the H291A-alpha mutation results in a complete order-to-disorder transition of the loop region, which precludes the binding of the substrate lipoyl-bearing domain to E1b. The H146A-beta' mutation, on the other hand, does not alter the loop structure, but nullifies the reductive acylation activity of E1b. Our results suggest that: 1) His291-alpha plays a structural rather than a catalytic role in the binding of cofactor ThDP and the lipoyl-bearing domain to E1b, and 2) His146-beta' is an essential catalytic residue, probably functioning as a proton donor in the reductive acylation of lipoamide on the lipoyl-bearing domain.     

Crystal structure of a 12 ANK repeat stack from human ankyrinR

Ankyrins are multifunctional adaptors that link specific proteins to the membrane-associated, spectrin- actin cytoskeleton. The N-terminal, 'membrane-binding' domain of ankyrins contains 24 ANK repeats and mediates most binding activities. Repeats 13-24 are especially active, with known sites of interaction for the Na/K ATPase, Cl/HCO(3) anion exchanger, voltage-gated sodium channel, clathrin heavy chain and L1 family cell adhesion molecules. Here we report the crystal structure of a human ankyrinR construct containing ANK repeats 13-24 and a portion of the spectrin-binding domain. The ANK repeats are observed to form a contiguous spiral stack with which the spectrin-binding domain fragment associates as an extended strand. The structural information has been used to construct models of all 24 repeats of the membrane-binding domain as well as the interactions of the repeats with the Cl/HCO(3) anion exchanger and clathrin. These models, together with available binding studies, suggest that ion transporters such as the anion exchanger associate in a large central cavity formed by the ANK repeat spiral, while clathrin and cell adhesion molecules associate with specific regions outside this cavity.     

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.     

Crystal structure of the RIM2 C2A-domain at 1.4 A resolution

RIMs are large proteins that contain two C2-domains and are localized at presynaptic active zones, where neurotransmitters are released. RIMs play key roles in synaptic vesicle priming and regulation of presynaptic plasticity. A mutation in the RIM1 C2A-domain has been implicated in autosomal dominant cone-rod dystrophy (CORD7). The RIM C2A-domain does not contain the full complement of aspartate residues that commonly mediate Ca2+ binding at the top loops of C2-domains, and has been reported to interact with SNAP-25 and synaptotagmin 1, two proteins from the Ca2+-dependent membrane fusion machinery. Here we have used NMR spectroscopy and X-ray crystallography to analyze the structure and biochemical properties of the RIM2 C2A-domain, which is closely related to the RIM1 C2A-domain. We find that the RIM2 C2A-domain does not bind Ca2+. Moreover, little binding of the RIM2 C2A-domain to SNAP-25 and to the C2-domains of synaptotagmin 1 was detected by NMR experiments, suggesting that as yet unidentified interactions of the RIM C2A-domain mediate its function. The crystal structure of the RIM2 C2A-domain using data to 1.4 A resolution reveals a beta-sandwich that resembles those observed for other C2-domains, but exhibits a unique dipolar distribution of electrostatic charges whereby one edge of the beta-sandwich is highly positive and the other edge is highly negative. The location of the mutation site implicated in CORD7 at the bottom of the domain and the pattern of sequence conservation suggest that, in contrast to most C2-domains, the RIM C2A-domains may function through Ca2+-independent interactions involving their bottom face.