A biochemical and structural characterization of Drosophila neuroglian

A number of cell-cell interactions in the nervous system are mediated by immunoglobulin gene superfamily members. For example, neuroglian, a homophilic neural cell adhesion molecule in Drosophila, has an extracellular portion comprising six C- 2 type immunoglobulin-like domains followed by five fibronectin type III (FnIII) repeats. Neuroglian shares this domain organization and significant sequence identity with Ll, a murine neural adhesion molecule that could be a functional homologue. Here I report the crystal structure of a proteolytic fragment containing the first two FnIII repeats of neuroglian (NgFn 1,2) at 2.0Å. The interpretation of photomicrographs of rotary shadowed Ng, the entire extracellular portion of neuroglian, and NgFnl-5, the five neuroglian Fn III domains, is also discussed.

The structure of NgFn 1,2 consists of two roughly cylindrical β-barrel structural motifs arranged in a head-to-tail fashion with the domains meeting at an angle of ~120, as defined by the cylinder axes. The folding topology of each domain is identical to that previously observed for single FnIII domains from tenascin and fibronectin. The domains of NgFn1,2 are related by an approximate two fold screw axis that is nearly parallel to the longest dimension of the fragment. Assuming this relative orientation is a general property of tandem FnIII repeats, the multiple tandem FnIII domains in neuroglian and other proteins are modeled as thin straight rods with two domain zig-zag repeats. When combined with the dimensions of pairs of tandem immunoglobulin-like domains from CD4 and CD2, this model suggests that neuroglian is a long narrow molecule (20 - 30 Å in diameter) that extends up to 370Å from the cell surface.

In photomicrographs, rotary shadowed Ng and NgFn1-5 appear to be highly flexible rod-like molecules. NgFn 1-5 is observed to bend in at least two positions and has a mean total length consistent with models generated from the NgFn1,2 structure. Ng molecules have up to four bends and a mean total length of 392 Å, consistent with a head-to-tail packing of neuroglian's C2-type domains.

The LIN-3 gene of the nematode C. elegans is the vulval-inducing signal

The development of the vulva of the nematode Caenorhabditis elegans is induced by a signal from the anchor cell of the somatic gonad. Activity of the gene lin-3 is required for the Vulval Precursor Cells (VPCs) to assume vulval fates. It is shown here that lin-3 encodes the vulval-inducing signal.

lin-3 was molecularly cloned by transposon-tagging and shown to encode a nematode member ofthe Epidermal Growth Factor (EGF) family. Genetic epistasis experiments indicate that lin-3 acts upstream of let-23, which encodes a homologue of the EGF-Receptor.

lin-3 transgenes that contain multiple copies of wild-type lin-3 genomic DNA clones confer a dominant multivulva phenotype in which up to all six of the VPCs assume vulval fates. The properties of these trans genes suggest that lin-3 can act in the anchor cell to induce vulval fates. Ablation of the gonadal precursors, which prevents the development of the AC, strongly reduces the ability of lin-3 transgenes to stimulate vulval development. A lin-3 recorder transgene that retains the ability to stimulate vulval development is expressed specifically in the anchor cell at the time of vulval induction.

Expression of an obligate secreted form of the EGF domain of Lin-S from a heterologous promoter is sufficient to induce vulval fates in the absence of the normal source of the inductive signal. This result suggests that Lin-S may act as a secreted factor, and that Lin-S may be the sole vulval-inducing signal made by the anchor cell.

lin-3 transgenes can cause adjacent VPCs to assume the 1° vulval fate and thus can override the action of the lateral signal mediated by lin-12 that normally prevents adjacent 1° fates. This indicates that the production of Lin-3 by the anchor cell must be limited to allow the VPCs to assume the proper pattern of fates of so 3° 3° 2° 1° 2° 3°.

Structural requirements for catalysis: studies on RTEM-1 β-lactamase

β-lactamases are a group of enzymes that confer resistance to penam and cephem antibiotics by hydrolysis of the β-lactam ring, thereby inactivating the antibiotic. Crystallographic and computer modeling studies of RTEM-1 β-lactamase have indicated that Asp 132, a strictly conserved residue among the class A β-lactamases, appears to be involved in substrate binding, catalysis, or both. To study the contribution of residue 132 to β-lactamase function, site saturation mutagenesis was used to generate mutants coding for all 20 amino acids at position 132. Phenotypic screening of all mutants indicated that position 132 is very sensitive to amino acid changes, with only N132C, N132D, N132E, and N132Q showing any appreciable activity. Kinetic analysis of three of these mutants showed increases in K_M, along with substantial decreases in k_(cat). Efforts to trap a stable acyl-enzyme intermediate were unsuccessfuL These results indicate that residue 132 is involved in substrate binding, as well as catalysis, and supports the involvement of this residue in acylation as suggested by Strynadka et al.

Crystallographic and computer modeling studies of RTEM-1 β-lactamase have indicated that Lys 73 and Glu 166, two strictly conserved residues among the class A β-lactamases, appear to be involved in substrate binding, catalysis, or both. To study the contribution of these residues to β-lactamase function, site saturation mutagenesis was used to generate mutants coding for all 20 amino acids at positions 73 and 166. Then all 400 possible combinations of mutants were created by combinatorial mutagenesis. The colonies harboring the mutants were screened for growth in the presence of ampicillin. The competent colonys' DNA were sequenced, and kinetic parameters investigated. It was found that lysine is essential at position 73, and that position 166 only tolerated fairly conservative changes (Aspartic acid, Histidine, and Tyrosine). These functional mutants exhibited decreased kcat's, but K_M was close to wild-type levels. The results of the combinatorial mutagenesis experiments indicate that Lysis absolutely required for activity at position 73; no mutation at residue 166 can compensate for loss of the long side chain amine. The active mutants found--K73K/E166D, K73KIE166H, and K73KIE166Y were studied by kinetic analysis. These results reaffirmed the function of residue 166 as important in catalysis, specifically deacylation.

The identity of the residue responsible for enhancing the active site serine (Ser 70) in RTEM-1 β-lactamase has been disputed for some time. Recently, analysis of a crystal structure of RTEM-1 β-lactamase with covalently bound intermediate was published, and it was suggested that Lys 73, a strictly conserved residue among the class A β-lactamases, was acting as a general base, activating Ser 70. For this to be possible, the pK_a of Lys 73 would have to be depressed significantly. In an attempt to assay the pK_a of Lys 73, the mutation K73C was made. This mutant protein can be reacted with 2-bromoethylamine, and activity is restored to near wild type levels. ^(15)N-2-bromoethylamine hydrobromide and ^(13)C-2-bromoethylamine hydrobromide were synthesized. Reacting these compounds with the K73C mutant gives stable isotopic enrichment at residue 73 in the form of aminoethylcysteine, a lysine homologue. The pK_a of an amine can be determined by NMR titration, following the change in chemical shift of either the ^(15)N-amine nuclei or adjacent Be nuclei as pH is changed. Unfortunately, low protein solubility, along with probable label scrambling in the Be experiment, did not permit direct observation of either the ^(15)N or ^(13)C signals. Indirect detection experiments were used to observe the protons bonded directly to the ^(13)C atoms. Two NMR signals were seen, and their chemical shift change with pH variation was noted. The peak which was determined to correspond to the aminoethylcysteine residue shifted from 3.2 ppm down to 2.8 ppm over a pH range of 6.6 to 12.5. The pK_a of the amine at position 73 was determined to be ~10. This indicates that residue 73 does not function as a general base in the acylation step of the reaction. However the experimental measurement takes place in the absence of substrate. Since the enzyme undergoes conformational changes upon substrate binding, the measured pK_a of the free enzyme may not correspond to the pK_a of the enzyme substrate complex.

Characterization of the human SCF ubiquitin ligases - structure, function, and regulation

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by cata lyzi ng ubiquitination of the S phase CDK inhibitor SIC1. SCF is composed of several evolutionarily conserved proteins, including ySKP1, CDC53 (Cullin), and the F-box protein CDC4. We isolated hSKP1 in a two-hybrid screen with hCUL1, the human homologue of CDC53. We showed that hCUL1 associates with hSKP1 in vivo and directly interacts with hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-Iike particle. Moreover, hCUL1 complements the growth defect of yeast CDC53^(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. These data demonstrated that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. However, purified human SCF complexes consisting of CUL1, SKP1, and SKP2 are inactive in vitro, suggesting that additional factors are required.

Subsequently, mammalian SCF ubiquitin ligases were shown to regulate various physiological processes by targeting important cellular regulators, like lĸBα, β-catenin, and p27, for ubiquitin-dependent proteolysis by the 26S proteasome. Little, however, is known about the regulation of various SCF complexes. By using sequential immunoaffinity purification and mass spectrometry, we identified proteins that interact with human SCF components SKP2 and CUL1 in vivo. Among them we identified two additional SCF subunits: HRT1, present in all SCF complexes, and CKS1, that binds to SKP2 and is likely to be a subunit of SCF5^(SKP2) complexes. Subsequent work by others demonstrated that these proteins are essential for SCF activity. We also discovered that COP9 Signalosome (CSN), previously described in plants as a suppressor of photomorphogenesis, associates with CUL1 and other SCF subunits in vivo. This interaction is evolutionarily conserved and is also observed with other Cullins, suggesting that all Cullin based ubiquitin ligases are regulated by CSN. CSN regulates Cullin Neddylation presumably through CSNS/JAB1, a stochiometric Signalosome subunit and a putative deneddylating enzyme. This work sheds light onto an intricate connection that exists between signal transduction pathways and protein degradation machinery inside the cell and sets stage for gaining further insights into regulation of protein degradation.