The structure of Mlc titration factor A (MtfA/YeeI) reveals a prototypical zinc metallopeptidase related to anthrax lethal factor

MtfA of Escherichia coli (formerly YeeI) was previously identified as a regulator of the phosphoenolpyruvate (PEP)-dependent:glucose phosphotransferase system. MtfA homolog proteins are highly conserved, especially among beta- and gammaproteobacteria. We determined the crystal structures of the full-length MtfA apoenzyme from Klebsiella pneumoniae and its complex with zinc (holoenzyme) at 2.2 and 1.95 Å, respectively. MtfA contains a conserved H¹⁴⁹E¹⁵⁰XXH¹⁵³+E²¹²+Y²⁰⁵ metallopeptidase motif. The presence of zinc in the active site induces significant conformational changes in the region around Tyr205 compared to the conformation of the apoenzyme. Additionally, the zinc-bound MtfA structure is in a self-inhibitory conformation where a region that was disordered in the unliganded structure is now observed in the active site and a nonproductive state of the enzyme is formed. MtfA is related to the catalytic domain of the anthrax lethal factor and the Mop protein involved in the virulence of Vibrio cholerae, with conservation in both overall structure and in the residues around the active site. These results clearly provide support for MtfA as a prototypical zinc metallopeptidase (gluzincin clan).     

A protocol for high-throughput phenotyping, suitable for quantitative trait analysis in mice

Whole-genome genetic association studies in outbred mouse populations represent a novel approach to identifying the molecular basis of naturally occurring genetic variants, the major source of quantitative variation between inbred strains of mice. Measuring multiple phenotypes in parallel on each mouse would make the approach cost effective, but protocols for phenotyping on a large enough scale have not been developed. In this article we describe the development and deployment of a protocol to collect measures on three models of human disease (anxiety, type II diabetes, and asthma) as well as measures of mouse blood biochemistry, immunology, and hematology. We report that the protocol delivers highly significant differences among the eight inbred strains (A/J, AKR/J, BALBc/J, CBA/J, C3H/HeJ, C57BL/6 J, DBA/2 J, and LP/J), the progenitors of a genetically heterogeneous stock (HS) of mice. We report the successful collection of multiple phenotypes from 2000 outbred HS animals. The phenotypes measured in the protocol form the basis of a large-scale investigation into the genetic basis of complex traits in mice designed to examine interactions between genes and between genes and environment, as well as the main effects of genetic variants on phenotypes.     

Insights into Substrate Specificity of Geranylgeranyl Reductases Revealed by the Structure of Digeranylgeranylglycerophospholipid Reductase, an Essential Enzyme in the Biosynthesis of Archaeal Membrane Lipids

Archaeal membrane lipids consist of branched, saturated hydrocarbons distinct from those found in bacteria and eukaryotes. Digeranylgeranylglycerophospholipid reductase (DGGR) catalyzes the hydrogenation process that converts unsaturated 2,3-di-O-geranylgeranylglyceryl phosphate to saturated 2,3-di-O-phytanylglyceryl phosphate as a critical step in the biosynthesis of archaeal membrane lipids. The saturation of hydrocarbon chains confers the ability to resist hydrolysis and oxidation and helps archaea withstand extreme conditions. DGGR is a member of the geranylgeranyl reductase family that is also widely distributed in bacteria and plants, where the family members are involved in the biosynthesis of photosynthetic pigments. We have determined the crystal structure of DGGR from the thermophilic heterotrophic archaea Thermoplasma acidophilum at 1.6 Å resolution, in complex with flavin adenine dinucleotide (FAD) and a bacterial lipid. The DGGR structure can be assigned to the well-studied, p-hydroxybenzoate hydroxylase (PHBH) SCOP superfamily of flavoproteins that include many aromatic hydroxylases and other enzymes with diverse functions. In the DGGR complex, FAD adopts the IN conformation (closed) previously observed in other PHBH flavoproteins. DGGR contains a large substrate-binding site that extends across the entire ligand-binding domain. Electron density corresponding to a bacterial lipid was found within this cavity. The cavity consists of a large opening that tapers down to two, narrow, curved tunnels that closely mimic the shape of the preferred substrate. We identified a sequence motif, PxxYxWxFP, that defines a specificity pocket in the enzyme and precisely aligns the double bond of the geranyl group with respect to the FAD cofactor, thus providing a structural basis for the substrate specificity of geranylgeranyl reductases. DGGR is likely to share a common mechanism with other PHBH enzymes in which FAD switches between two conformations that correspond to the reductive and oxidative half cycles. The structure provides evidence that substrate binding likely involves conformational changes, which are coupled to the two conformational states of the FAD.     

Digging and marble burying in mice: simple methods for in vivo identification of biological impacts

Mice exhibit various species-typical behaviors such as digging and burrowing. They dig in the ground to find food, to hoard food, to create a refuge from predators or cold and to make a safe nursery area for the young. In the laboratory, mice dig vigorously in deep bedding such as wood chips. This behavior is sensitive to strain differences and drugs. For example, the effects of anxiolytics and 5-HT-active compounds, including those used clinically for obsessive-compulsive disorder (OCD), can be detected. Digging can be quantified by manual timing. Alternatively, the bedding can be covered with glass marbles and the number buried can be counted after a set time. These behaviors can be assessed using very little specialized equipment, and results can be obtained from ten animals in about an hour. Species-typical behaviors may be sensitive to a wide variety of treatments, and their simplicity and ability to yield robust quantitative data might be particularly useful in assessing genetically modified mice, even in laboratories not primarily oriented to behavioral work.     

Assessing nest building in mice

For small rodents, nests are important in heat conservation as well as reproduction and shelter. Nesting is easily measured in the home cages of mice, particularly with the advent of pressed cotton materials. The mice first shred the tightly packed material, then arrange it into a nest. Published studies have often used materials such as hay, twine or tissues, sometimes preshredded, and have assigned scores of the quality of the resulting nest with rather rudimentary rating scales; e.g., 0, no nest; 1, flat nest; 2, nest covering the mouse. The protocol described here uses pressed cotton squares and a definitive 5-point nest-rating scale. Any unshredded material left after a bout of nesting can also be weighed, providing a semi-independent objective assay of nesting ability. Nesting has been shown to be sensitive to brain lesions, pharmacological agents and genetic mutations. This is a simple, cheap and easily done test that, along with other tests of species-typical behavior, is a sensitive assay for identifying previously unknown behavioral phenotypes. The test needs to be done overnight, but it should take no more than 5 minutes to set up plus 1 minute to assess one nest and weigh the untorn residue.     

Housing, husbandry and handling of rodents for behavioral experiments

Most animals used in research are rodents, mainly mice because of their predominance in genetics and molecular biology. This article attempts to provide an introduction to mice and rats: health considerations (of the experimenter); choice of species, age, strain and sex; housing and environmental enrichment; and animal identification, handling and dosing. These considerations apply to animal work in general; the rest of the article focuses on the preliminary aspects of behavioral testing, including a protocol for an open field test. This procedure is traditionally associated with activity measurements, and although automated versions are readily available these days, the latter are expensive and may be unavailable in many non-behavioral departments. Moreover, particularly when testing novel genetically modified animals or pharmacological agents, there is no substitute for direct visual observation to detect abnormal signs in the animals: for example, ptosis, piloerection, tremor, ataxia or exophthalmos. The open field test can be adapted in several ways: to assess general behavior and activity (similar to a primary screen in the pharmaceutical industry) or to measure memory (habituation) or anxiety.     

T-maze alternation in the rodent

This protocol details a method for using a T-maze to assess the cognitive ability of rodents. The T-maze is an elevated or enclosed apparatus in the form of a T placed horizontally. Animals are started from the base of the T and allowed to choose one of the goal arms abutting the other end of the stem. If two trials are given in quick succession, on the second trial the rodent tends to choose the arm not visited before, reflecting memory of the first choice. This is called 'spontaneous alternation'. This tendency can be reinforced by making the animal hungry and rewarding it with a preferred food if it alternates. Both spontaneous and rewarded alternation are very sensitive to dysfunction of the hippocampus, but other brain structures are also involved. Each trial should be completed in under 2 min, but the total number of trials required will vary according to statistical and scientific requirements.