ClpP is a self-compartmentalized proteolytic assembly comprised of two, stacked, heptameric rings that, when associated with its cognate hexameric ATPase (ClpA or ClpX), form the ClpAP and ClpXP ATP-dependent protease, respectively. The symmetry mismatch is an absolute feature of this large energy-dependent protease and also of the proteasome, which shares a similar barrelshaped architecture, but how it is accommodated within the complex has yet to be understood, despite recent structural investigations, due in part to the conformational lability of the N-termini. We present the structures of Escherichia coli ClpP to 1.9 Å and an inactive variant that provide some clues for how this might be achieved. In the wild type protein, the highly conserved Nterminal 20 residues can be grouped into two major structural classes. In the first, a loop formed by residues 10-15 protrudes out of the central access channel extending ∼ 12-15 Å from the surface of the oligomer resulting in the closing of the access channel observed in one ring. Similar loops are implied to be exclusively observed in human ClpP and a variant of ClpP from Streptococcus pneumoniae. In the other ring, a second class of loop is visible in the structure of wt ClpP from E. coli that forms closer to residue 16 and faces toward the interior of the molecule creating an open conformation of the access channel. In both classes, residues 18-20 provide a conserved interaction surface. In the inactive variant, a third class of N-terminal conformation is observed, which arises from a conformational change in the position of F17. We have performed a detailed functional analysis on each of the first 20 amino acid residues of ClpP. Residues that extend beyond the plane of the molecule (10-15) have a lesser effect on ATPase interaction than those lining the pore (1-7 and 16-20). Based upon our structure-function analysis, we present a model to explain the widely disparate effects of individual residues on ClpP-ATPase complex formation and also a possible functional reason for this mismatch.
Maple syrup urine disease (MSUD) is an inherited disorder of branched-chain amino acid metabolism presenting with life-threatening cerebral oedema and dysmyelination in affected individuals. Treatment requires life-long dietary restriction and monitoring of branched-chain amino acids to avoid brain injury. Despite careful management, children commonly suffer metabolic decompensation in the context of catabolic stress associated with non-specific illness. The mechanisms underlying this decompensation and brain injury are poorly understood. Using recently developed mouse models of classic and intermediate maple syrup urine disease, we assessed biochemical, behavioural and neuropathological changes that occurred during encephalopathy in these mice. Here, we show that rapid brain leucine accumulation displaces other essential amino acids resulting in neurotransmitter depletion and disruption of normal brain growth and development. A novel approach of administering norleucine to heterozygous mothers of classic maple syrup urine disease pups reduced branched-chain amino acid accumulation in milk as well as blood and brain of these pups to enhance survival. Similarly, norleucine substantially delayed encephalopathy in intermediate maple syrup urine disease mice placed on a high protein diet that mimics the catabolic stress shown to cause encephalopathy in human maple syrup urine disease. Current findings suggest two converging mechanisms of brain injury in maple syrup urine disease including: (i) neurotransmitter deficiencies and growth restriction associated with branched-chain amino acid accumulation and (ii) energy deprivation through Krebs cycle disruption associated with branched-chain ketoacid accumulation. Both classic and intermediate models appear to be useful to study the mechanism of brain injury and potential treatment strategies for maple syrup urine disease. Norleucine should be further tested as a potential treatment to prevent encephalopathy in children with maple syrup urine disease during catabolic stress.
We characterized nine helicase-deficient mutants of bacteriophage T7 helicase-primase protein (4A) prepared by random mutagenesis as reported in the accompanying paper (Rosenberg, DNA helicases catalyze unwinding of duplex DNA to singlestranded DNA, a process energetically coupled to NTP hydrolysis. Helicases are an important class of proteins required in almost all the processes of DNA and RNA metabolism. Recently, a large number of putative helicases have been identified mainly from amino acid sequence homologies. Known helicases have homologous amino acid sequences, confined to small regions in the protein, that are used as signature motifs for identifying helicases (1, 2). Since a high resolution structure of a helicase is not known at the present time, the roles of these conserved motifs remain largely unclear.AWe are studying the mechanism of bacteriophage T7 DNA helicase, which is involved in DNA replication. Bacteriophage T7 is a model system used to study the detailed mechanisms of DNA replication because of its simplicity. A minimum of two proteins, T7 DNA polymerase and T7 DNA primase/helicase, have been shown to reconstitute duplex DNA replication in vitro. T7 gene 4 encodes the two primase/helicase proteins, 4A and 4B (3). The full-length 63-kDa 4A protein has both helicase and primase activities, whereas the shorter 56-kDa 4B protein that begins at a second initiation codon has only helicase activity (4, 5). The helicase activity unwinds double-stranded DNA during leading strand DNA replication, and the primase catalyzes synthesis of tetraribonucleotides that serve as primers for lagging strand DNA replication (4).T7 DNA helicase belongs to the general class of hexameric helicases. Its low resolution structure, studied in detail using electron microscopy and image averaging, shows that both 4AЈ and 4B proteins form ring-shaped hexamers, and the ssDNA 1 binds through the central hole of the ring (6). This mode of DNA binding results in protection of about 25 bases of ssDNA from nuclease digestion (7) and likely confers high processivity to DNA unwinding. The helicase forms hexamers only in the presence of nucleotide ligands such as dTDP, dTTP, ATP, and dTMP-PCP (8, 9). DNA binds tightly only to the hexameric species and requires the presence of dTTP or dTMP-PCP (7). The various activities of the helicase protein such as NTP binding/hydrolysis, protein oligomerization, and DNA binding are linked (9). Therefore, it is likely that amino acids responsible for these activities also may be close in space or perhaps lie in the same motif.Regions of T7 gene 4 protein show sequence homology to several bacterial and bacteriophage primase/helicase and primase-related helicases that belong to the DnaB family of helicases. Comparison of amino acid sequences in this family of helicases has led to the identification of five conserved motifs denoted 1A, 1a, 2B, 3, and 4 (2). Conserved motif 1A is the well known GXXGXGKT/S sequence found in numerous nucleotidebinding proteins and shown in many ATPases to be involved...
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