Dimeric structure of the six-domain VibF subunit of vibriobactin synthetase: mutant domain activity regain and ultracentrifugation studies

Nonribosomal peptide synthetases (NRPS), fatty acid synthases (FAS), and polyketide sythases (PKS) are multimodular enzymatic assembly lines utilized in natural product biosynthesis. Previous data on FAS and PKS subunits have indicated that they are homodimers and that some of their catalytic functions can work in trans. When NRPS assembly lines have been probed for comparable formation of stable oligomers, no evidence had been forthcoming that species other than monomer forms were active. In this work we focus on the six-domain (Cy1-Cy2-A-C1-PCP-C2) enzyme VibF from the vibriobactin synthetase assembly line, which contains three other proteins, VibB, VibE, and VibH, that--when purified and mixed with VibF and the substrates ATP, threonine, 2,3-dihydroxybenzoate (DHB), and norspermidine--produce the iron chelator vibriobactin. Using a deletion of the Cy1 domain and separate inactivating mutations in the Cy2, A, PCP, and C2 domains of VibF, we report regain of catalytic activity upon mutant protein mixing that argues for heterodimer formation, stable for hundreds to thousands of catalytic cycles, with acyl chain processing and transfer around blocked domains. Ultracentrifugation data likewise confirm a dimeric structure for VibF and establish that domains within NRPS dimeric modules can act on acyl chains in trans. The results described here are the first indication for an NRPS subunit that homodimerization can occur and that there is a continuum of functional oligomerization states between monomers and dimers in nonribosomal peptide synthetases.     

Direct mass spectrometric determination of the stoichiometry and binding affinity of the complexes between nucleocapsid protein and RNA stem-loop hairpins of the HIV-1 Psi-recognition element

The formation of noncovalent complexes between the HIV-1 nucleocapsid protein p7 (NC) and RNA hairpins SL2-SL4 of the Psi-recognition element was investigated by direct infusion electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The high resolution afforded by this method provided the unambiguous characterization of the stoichiometry and composition of complexes formed by multiple equilibria in solution. For each hairpin, the formation of a 1:1 complex was found to be the primary binding mode in solutions of intermediate salt content (150 mM ammonium acetate). Binding of multiple units of NC was observed with lower affinity and a maximum stoichiometry matching the limit calculated from the number of nucleotides in the construct and the size of the footprint of NC onto single-stranded nucleic acids, thus implying the defolding of the hairpin three-dimensional (3D) structure. Dissociation constants of 62 +/- 22 nM, 178 +/- 64 nM, and 1.3 +/- 0.5 microM were determined for SL2, SL3-2, and SL4, respectively, which are similar to values obtained by spectroscopic and calorimetric methods with the additional confidence offered by a direct, rather than inferred, knowledge of the binding stoichiometry. Competitive binding experiments carried out in solutions of intermediate ionic strength, which has the effect of weakening the electrostatic interactions in solution, provided a direct way of evaluating the stabilizing contributions of H-bonding and hydrophobic interactions that are more sensitive to the sequence and structural context of the different hairpins. The relative scale of binding affinity obtained in this environment reflects the combination of contributions provided by the different structures of both the tetraloop and the double-stranded stem. The importance of the stem 3D structure in modulating the binding activity was tested by a competitive binding experiment that included the SL3-2 RNA construct, a DNA analogue of SL3 (SL3(DNA)), and a DNA analogue in which all four loop bases were replaced with abasic nucleotides (SL3(abasic)). NC was found to bind the A-type double-stranded stem of SL3-2 RNA at least 30 times more tightly than the B-type helical structure of SL3(DNA). Eliminating the stabilization provided by the interactions with the tetraloop bases made the binding of SL3(abasic) approximately 50 times weaker than that of SL3(DNA).     

Molecular dynamics simulations of Trp side-chain conformational flexibility in the gramicidin A channel

Gramicidin A (gA) is prototypical peptide antibiotic and a model ion channel former. Configured in the solid-state NMR beta(6.5)-helix channel conformation, gA was subjected to 1-ns molecular dynamics (MD) gas phase simulations using the all-atom charmm22 force field to ascertain the conformational stability of the Trp side chains as governed by backbone and neighboring side-chain contacts. Three microcanonical trajectories were computed using different initial atomic velocities for each of twenty different initial structures. For each set, one of the four Trp side chains in each monomer was initially positioned in one of the five non-native conformations (A. E. Dorigo et al., Biophysical Journal, 1999, Vol. 76, 1897-1908), the other Trps being positioned in the native state, o1. In three additional control simulations, all Trps were initiated in the native conformation. After equilibration, constraints were removed and subsequent conformational changes of the initially constrained Trp were measured. The chi(1) was more flexible than chi(2.1). The energetically optimal orientation, o1 (Dorigo et al., 1999), was the most stable in all four Trp positions (9, 11, 13, 15) and remained unchanged for the entire 1 ns simulation in 19 of 24 trials. Changes in chi(1) from each of the 5 suboptimal states occur readily. Two of the non-native conformations reverted readily to o1, whereas the other three converted to an intermediate state, i2. There were frequent interconversions between i2 and o1. We speculate that experimentally observed Trp stability is caused by interactions with the lipid-water interface, and that stabilization of one of the suboptimal conformations in gA, such as i2, by lipid headgroups could produce a secondary, metastable conformational state. This could explain recent experimental studies of differences in the channel conductance dispersity between gA and a Trp-to-Phe gA analog, gramicidin M (gM, J. C. Markham et al., Biochimica et Biophysica Acta, 2001, Vol. 1513, 185-192).     

Microarray analysis of murine palatogenesis: temporal expression of genes during normal palate development

The mammalian face is assembled in utero in a series of complex and interdependent molecular, cell and tissue processes. The orofacial complex appears to be exquisitely sensitive to genetic and environmental influence and this explains why clefts of the lip and palate are the most common congenital anomaly in humans (one in 700 live births). In this study, microarray technology was used to identify genes that may play pivotal roles in normal murine palatogenesis. mRNA was isolated from murine embryonic palatal shelves oriented vertically (before elevation), horizontally (following elevation, before contact), and following fusion. Changes in gene expression between the three different stages were analyzed with GeneChip microarrays. A number of genes were upregulated or downregulated, and large changes were seen in the expression of loricrin, glutamate decarboxylase, gamma-amino butyric acid type A receptor beta3 subunit, frizzled, Wnt-5a, metallothionein, annexin VIII, LIM proteins, Sox1, plakophilin1, cathepsin K and creatine kinase. In this paper, the changes in genetic profile of the developing murine palate are presented, and the possible role individual genes/proteins may play during normal palate development are discussed. Candidate genes with a putative role in cleft palate are also highlighted.     

A 65-kilobase pathogenicity island is unique to Philadelphia-1 strains of Legionella pneumophila

Nucleotide sequence analysis of an approximately 80-kb genomic region revealed an approximately 65-kb locus that bears hallmarks of a pathogenicity island. This locus includes homologues of a type IV secretion system, mobile genetic elements, and known virulence factors. Comparative studies with other Legionella pneumophila strains and serogroups indicated that this approximately 65-kb locus is unique to L. pneumophila serogroup 1 Philadelphia-1 strains.     

Crystal structures of the BtuF periplasmic-binding protein for vitamin B12 suggest a functionally important reduction in protein mobility upon ligand binding

BtuF is the periplasmic binding protein (PBP) for the vitamin B12 transporter BtuCD, a member of the ATP-binding cassette (ABC) transporter superfamily of transmembrane pumps. We have determined crystal structures of Escherichia coli BtuF in the apo state at 3.0 A resolution and with vitamin B12 bound at 2.0 A resolution. The structure of BtuF is similar to that of the FhuD and TroA PBPs and is composed of two alpha/beta domains linked by a rigid alpha-helix. B12 is bound in the "base-on" or vitamin conformation in a wide acidic cleft located between these domains. The C-terminal domain shares structural homology to a B12-binding domain found in a variety of enzymes. The same surface of this domain interacts with opposite surfaces of B12 when comparing ligand-bound structures of BtuF and the homologous enzymes, a change that is probably caused by the obstruction of the face that typically interacts with this domain by the base-on conformation of vitamin B12 bound to BtuF. There is no apparent pseudo-symmetry in the surface properties of the BtuF domains flanking its B12 binding site even though the presumed transport site in the previously reported crystal structure of BtuCD is located in an intersubunit interface with 2-fold symmetry. Unwinding of an alpha-helix in the C-terminal domain of BtuF appears to be part of conformational change involving a general increase in the mobility of this domain in the apo structure compared with the B12-bound structure. As this helix is located on the surface likely to interact with BtuC, unwinding of the helix upon binding to BtuC could play a role in triggering release of B12 into the transport cavity. Furthermore, the high mobility of this domain in free BtuF could provide an entropic driving force for the subsequent release of BtuF required to complete the transport cycle.     

Interval timing in rats: tracking unsignaled changes in the fixed interval schedule requirement

The present experiment examined interval timing in rats under dynamic conditions. A session began with FI60 s intervals, changed to a FI20 s, FI30 s, or FI40 s schedule at an unpredictable point, and then returned to a FI60 s schedule after the rats received 1, 8, or 24 successive short FI intervals. Variations in the duration and number of shorter intervals occurred across sessions and conditions. We observed rapid control of wait time duration by the FI duration of the preceding interval (one-back tracking), and changes in wait time depended on the number and duration of the shorter intervals. Furthermore, we observed proportional and scalar timing effects in overall wait time duration. The results provide information about the relation between interval timing under dynamic and steady state conditions.     

Overexpression of murine phosphatidylinositol 4-phosphate 5-kinase type Ibeta disrupts a phosphatidylinositol 4,5 bisphosphate regulated endosomal pathway

The type I phosphatidylinositol 4-phosphate 5-kinases (PI4P5K) phosphorylate phosphatidylinositol 4-phosphate [PI(4)P] to produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 has been implicated in signal transduction, receptor mediated endocytosis, vesicle trafficking, cytoskeletal structure, and membrane ruffling. However, the specific type I enzymes associated with the production of PI(4,5)P2 for the specific cellular processes have not been rigorously defined. Murine PI4P5K type Ibeta (mPIP5K-Ibeta) was implicated in receptor mediated endocytosis through the isolation of a truncated and inactive form of the enzyme that blocked the ligand-dependent downregulation of the colony-stimulating factor-1 receptor. The present study shows that enforced expression of mPIP5K-Ibeta in 293T cells resulted in the accumulation of large vesicles that were linked to an endosomal pathway. Similar results were obtained after the expression of the PI(4,5)P2-binding pleckstrin homology (PH) domain of phospholipase-Cdelta (PLC-delta). Analysis of the conserved domains of mPIP5K-Ibeta led to the identification of dimerization domains in the N- and C-terminal regions. Enforced expression of the individual dimerization domains interfered with the proper subcellular localization of mPIP5K-Ibeta and the PLC-delta-PH domain and blocked the accumulation of the endocytic vesicles induced by these proteins. In addition to regulating early steps in endocytosis, these results suggest that mPIP5K-Ibeta acts through PI(4,5)P2 to regulate endosomal trafficking and/or fusion.     

Multiple ribonuclease H-encoding genes in the Caenorhabditis elegans genome contrasts with the two typical ribonuclease H-encoding genes in the human genome

Database searches of the Caenorhabditis elegans and human genomic DNA sequences revealed genes encoding ribonuclease H1 (RNase H1) and RNase H2 in each genome. The human genome contains a single copy of each gene, whereas C. elegans has four genes encoding RNase H1-related proteins and one gene for RNase H2. By analyzing the mRNAs produced from the C. elegans genes, examining the amino acid sequence of the predicted protein, and expressing the proteins in Esherichia coli we have identified two active RNase H1-like proteins. One is similar to other eukaryotic RNases H1, whereas the second RNase H (rnh-1.1) is unique. The rnh-1.0 gene is transcribed as a dicistronic message with three dsRNA-binding domains; the mature mRNA is transspliced with SL2 splice leader and contains only one dsRNA-binding domain. Formation of RNase H1 is further regulated by differential cis-splicing events. A single rnh-2 gene, encoding a protein similar to several other eukaryotic RNase H2L's, also has been examined. The diversity and enzymatic properties of RNase H homologues are other examples of expansion of protein families in C. elegans. The presence of two RNases H1 in C. elegans suggests that two enzymes are required in this rather simple organism to perform the functions that are accomplished by a single enzyme in more complex organisms. Phylogenetic analysis indicates that the active C. elegans RNases H1 are distantly related to one another and that the C. elegans RNase H1 is more closely related to the human RNase H1. The database searches also suggest that RNase H domains of LTR-retrotransposons in C. elegans are quite unrelated to cellular RNases H1, but numerous RNase H domains of human endogenous retroviruses are more closely related to cellular RNases H.