Saccharomyces cerevisiae SMT4 encodes an evolutionarily conserved protease with a role in chromosome condensation regulation

In a search for regulatory genes affecting the targeting of the condensin complex to chromatin in Saccharomyces cerevisiae, we identified a member of the adenovirus protease family, SMT4. SMT4 overexpression suppresses the temperature-sensitive conditional lethal phenotype of smc2-6, but not smc2-8 or smc4-1. A disruption allele of SMT4 has a prominent chromosome phenotype: impaired targeting of Smc4p-GFP to rDNA chromatin. Site-specific mutagenesis of the predicted protease active site cysteine and histidine residues of Smt4p abolishes the SMT4 function in vivo. The previously uncharacterized SIZ1 (SAP and Miz) gene, which encodes a protein containing a predicted DNA-binding SAP module and a Miz finger, is identified as a bypass suppressor of the growth defect associated with the SMT4 disruption. The SIZ1 gene disruption is synthetically lethal with the SIZ2 deletion. We propose that SMT4, SIZ1, and SIZ2 are involved in a novel pathway of chromosome maintenance.     

Solution 1H NMR of the active site of substrate-bound, cyanide-inhibited human heme oxygenase. comparison to the crystal structure of the water-ligated form

The majority of the active site residues of cyanide-inhibited, substrate-bound human heme oxygenase have been assigned on the basis of two-dimensional NMR using the crystal structure of the water-ligated substrate complex as a guide (Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Nat. Struct. Biol. 6, 860-867). The proximal helix and the N-terminal portion of the distal helix are found to be identical to those in the crystal except that the heme for the major isomer ( approximately 75-80%) in solution is rotated 180 degrees about the alpha-gamma-meso axis relative to the unique orientation in the crystal. The central portion of the distal helix in solution is translated slightly over the heme toward the distal ligand, and a distal four-ring aromatic cluster has moved 1-2 A closer to the heme, which allows for strong hydrogen bonds between the hydroxyls of Tyr-58 and Tyr-137. These latter interactions are proposed to stabilize the closed pocket conducive to the high stereospecificity of the alpha-meso ring opening. The determination of the magnetic axes, for which the major axis is controlled by the Fe-CN orientation, reveals a approximately 20 degrees tilt of the distal ligand from the heme normal in the direction of the alpha-meso bridge, demonstrating that the close placement of the distal helix over the heme exerts control of stereospecificity by both blocking access to the beta, gamma, and delta-meso positions and tilting the axial ligand, a proposed peroxide, toward the alpha-meso position.     

THUMP--a predicted RNA-binding domain shared by 4-thiouridine, pseudouridine synthases and RNA methylases

Sequence profile searches were used to identify an ancient domain in ThiI-like thiouridine synthases, conserved RNA methylases, archaeal pseudouridine synthases and several uncharacterized proteins. We predict that this domain is an RNA-binding domain that adopts an alpha/beta fold similar to that found in the C-terminal domain of translation initiation factor 3 and ribosomal protein S8.     

A bipolar DNA helicase gene, herA, clusters with rad50, mre11 and nurA genes in thermophilic archaea

We showed previously that rad50 and mre11 genes of thermophilic archaea are organized in an operon-like structure with a third gene (nurA) encoding a 5' to 3' exonuclease. Here, we show that the rad50, mre11 and nurA genes from the hyperthermophilic archaeon Sulfolobus acidocaldarius are co-transcribed with a fourth gene encoding a DNA helicase. This enzyme (HerA) is the prototype of a new class of DNA helicases able to utilize either 3' or 5' single-stranded DNA extensions for loading and subsequent DNA duplex unwinding. To our knowledge, DNA helicases capable of translocating along the DNA in both directions have not been identified previously. Sequence analysis of HerA shows that it is a member of the TrwB, FtsK and VirB4/VirD4 families of the PilT class NTPases. HerA homologs are found in all thermophilic archaeal species and, in all cases except one, the rad50, mre11, nurA and herA genes are grouped together. These results suggest that the archaeal Rad50-Mre11 complex might act in association with a 5' to 3' exonuclease (NurA) and a bipolar DNA helicase (HerA) indicating a probable involvement in the initiation step of homologous recombination.     

The emergence of catalytic and structural diversity within the beta-clip fold

The beta-clip fold includes a diverse group of protein domains that are unified by the presence of two characteristic waist-like constrictions, which bound a central extended region. Members of this fold include enzymes like deoxyuridine triphosphatase and the SET methylase, carbohydrate-binding domains like the fish antifreeze proteins/Sialate synthase C-terminal domains, and functionally enigmatic accessory subunits of urease and molybdopterin biosynthesis protein MoeA. In this study, we reconstruct the evolutionary history of this fold using sensitive sequence and structure comparisons methods. Using sequence profile searches, we identified novel versions of the beta-clip fold in the bacterial flagellar chaperone FlgA and the related pilus protein CpaB, the StrU-like dehydrogenases, and the UxaA/GarD-like hexuronate dehydratases (SAF superfamily). We present evidence that these versions of the beta-clip domain, like the related type III anti-freeze proteins and C-terminal domains of sialic acid synthases, are involved in interactions with carbohydrates. We propose that the FlgA and CpaB-like proteins mediate the assembly of bacterial flagella and Flp pili by means of their interactions with the carbohydrate moieties of peptidoglycan. The N-terminal beta-clip domain of the hexuronate dehydratases appears to have evolved a novel metal-binding site, while their C-terminal domain is likely to adopt a metal-binding TIM barrel-like fold. Using structural comparisons, we show that the beta-clip fold can be further classified into two major groups, one that includes the SAF, SET, dUTPase superfamilies, and the other that includes the phage lambda head decoration protein, the beta subunit of urease and the C-terminal domain of the molybdenum cofactor biosynthesis protein MoeA. Structural comparisons also suggest the beta-clip fold was assembled through the duplication of a three-stranded unit. Though the three-stranded units are likely to have had a common origin, we present evidence that complete beta-clip domains were assembled through such duplications, independently on multiple occasions. There is also evidence for circular permutation of the basic three-stranded unit on different occasions in the evolution of the beta-clip unit. We also describe how assembly of this fold from a basic three-stranded unit has been utilized to accommodate a variety of activities in its different versions.     

Oxidation of Met144 and Met145 in calmodulin blocks calmodulin dependent activation of the plasma membrane Ca-ATPase

Methionine oxidation in calmodulin (CaM) isolated from senescent brain results in an inability to fully activate the plasma membrane (PM) Ca-ATPase, which may contribute to observed increases in cytosolic calcium levels under conditions of oxidative stress and biological aging. To identify the functional importance of the oxidation of Met(144) and Met(145) near the carboxyl-terminus of CaM, we have used site-directed mutagenesis to substitute leucines for methionines at other positions in CaM, permitting the site-specific oxidation of Met(144) and Met(145). Prior to their oxidation, the CaM-dependent activation of the PM-Ca-ATPase by these CaM mutants is similar to that of wild-type CaM. Likewise, oxidation of individual methionines has a minimal effect on the CaM concentration necessary for half-maximal activation of the PM-Ca-ATPase. These results are consistent with previous suggestions that no single methionine within CaM is essential for activation of the PM-Ca-ATPase. Oxidation of either Met(144) and Met(145) or all nine methionines in CaM results in an equivalent inhibition of the PM-Ca-ATPase, resulting in a 50-60% reduction in the level of enzyme activation. Oxidation of Met(144) is largely responsible for the decreased extent of enzyme activation, suggesting that this site is critical in modulating the sensitivity of CaM to oxidant-induced loss-of-function. These results are discussed in terms of a possible functional role for Met(144) and Met(145) in CaM as redox sensors that function to modulate calcium homeostasis and energy metabolism in response to conditions of oxidative stress.     

Cytosolic delivery of macromolecules: I. Synthesis and characterization of pH-sensitive acyloxyalkylimidazoles

A series of 1-(acyloxyalkyl)imidazoles (AAI) were synthesized by nucleophilic substitution of chloroalkyl esters of fatty acids with imidazole. The former was prepared from fatty acid chloride and an aldehyde. When incorporated into liposomes, these lipids show an apparent pK(a) value ranging from 5.12 for 1-(palmitoyloxymethyl)imidazole (PMI) to 5.29 for 1-[(alpha-myristoyloxy)ethyl]imidazole (alpha-MEI) as determined by a fluorescence assay. When the imidazole moiety was protonated, the lipids were surface-active, as demonstrated by hemolytic activity towards red blood cells. As expected, AAI were hydrolyzed in serum as well as in cell homogenate. They were significantly less toxic than biochemically stable N-dodecylimidazole (NDI) towards Chinese hamster ovary (CHO) and RAW 264.7 (RAW) cells as determined by MTT assay. When fed to RAW cells, fluorescein-labeled oligonucleotides encapsulated in liposomes containing 20 mol% 1-(stearoyloxymethyl)imidazole (SMI) resulted in punctate as well as partially diffuse fluorescence. In a functional assay involving down-regulation of luciferase in CV-1 cells, neutral liposomes containing imidazole lipids showed suboptimal delivery of antisense phosphorothioate oligomers. Taken together, the results suggest that AAI are of potential use in developing nontoxic, pH-sensitive liposomes. However, these liposomal formulations need to be optimized to achieve higher concentrations of pH-sensitive detergents within the endosome to facilitate efficient cytosolic release of liposome-entrapped contents.     

Evolution and classification of P-loop kinases and related proteins

Sequences and structures of all P-loop-fold proteins were compared with the aim of reconstructing the principal events in the evolution of P-loop-containing kinases. It is shown that kinases and some related proteins comprise a monophyletic assemblage within the P-loop NTPase fold. An evolutionary classification of these proteins was developed using standard phylogenetic methods, analysis of shared sequence and structural signatures, and similarity-based clustering. This analysis resulted in the identification of approximately 40 distinct protein families within the P-loop kinase class. Most of these enzymes phosphorylate nucleosides and nucleotides, as well as sugars, coenzyme precursors, adenosine 5'-phosphosulfate and polynucleotides. In addition, the class includes sulfotransferases, amide bond ligases, pyrimidine and dihydrofolate reductases, and several other families of enzymes that have acquired new catalytic capabilities distinct from the ancestral kinase reaction. Our reconstruction of the early history of the P-loop NTPase fold includes the initial split into the common ancestor of the kinase and the GTPase classes, and the common ancestor of ATPases. This was followed by the divergence of the kinases, which primarily phosphorylated nucleoside monophosphates (NMP), but could have had broader specificity. We provide evidence for the presence of at least two to four distinct P-loop kinases, including distinct forms specific for dNMP and rNMP, and related enzymes in the last universal common ancestor of all extant life forms. Subsequent evolution of kinases seems to have been dominated by the emergence of new bacterial and, to a lesser extent, archaeal families. Some of these enzymes retained their kinase activity but evolved new substrate specificities, whereas others acquired new activities, such as sulfate transfer and reduction. Eukaryotes appear to have acquired most of their kinases via horizontal gene transfer from Bacteria, partly from the mitochondrial and chloroplast endosymbionts and partly at later stages of evolution. A distinct superfamily of kinases, which we designated DxTN after its sequence signature, appears to have evolved in selfish replicons, such as bacteriophages, and was subsequently widely recruited by eukaryotes for multiple functions related to nucleic acid processing and general metabolism. In the course of this analysis, several previously undetected groups of predicted kinases were identified, including widespread archaeo-eukaryotic and archaeal families. The results could serve as a framework for systematic experimental characterization of new biochemical and biological functions of kinases.