Marvin Gold

Structure-based design of a potent transition state analogue for TEM-1 β-lactamase

The structure of the plasmid-mediated β-lactamase TEM-1 has been solved in complex with a designed boronic acid inhibitor (1R)-1-acetamido-2-(3-carboxyphenyl)ethane boronic acid at 1.7 Å resolution. The boronate inhibitor was designed based on the crystallographic coordinates of the acyl-enzyme intermediate of TEM-1 bound to the substrate penicillin G. The boronate–TEM-1 complex is highly ordered and defines a novel transition state analogue of the deacylation step in the β-lactamase reaction pathway. The design principles of this highly effective inhibitor (Ki=110 nM) and the resulting structural and mechanistic implications are presented.

Studies on an in vitro system for the packaging and maturation of phage λ DNA

Abstract Packaging and maturation of λ DNA in cell-free extracts is arbitrarily partitioned into two stages. In stage 1, exogenously added, immature concatemeric λ DNA interacts with the A-gene product (pA) and phage proheads. After this reaction has occurred, a second extract is added to initiate a different set of events that results in phage production (stage 2). The use of highly purified pA and purified proheads during stage 1 results in a marked reduction in the final phage yield. The deficit is largely corrected by the addition of extracts of uninfected cells in stage 1. This observation suggests that one or more host factors are required in the packaging reaction. The testing in vitro for λ-specific requirements in stage 1 reveals that Nu1-negative extracts prepared from λt16-infected cells are phenotypically prohead positive, but pA negative, in the assay. This result indicates that the t16 mutation is in a locus that specifies a gene product. Complementation in vitro between λt16- and λAam-infected cell extracts has not been accomplished as yet. Paradoxically, highly purified pA can support optimal phage production in conjunction with Nu1-negative stage 1 and stage 2 extracts. Stage 2 requires the action of the products of λ genes D, W, and FII. Successful complementation among pairs of stage 2-deficient extracts provides convenient assays for purifying these proteins.

A novel in vitro DNA packaging system demonstrating a direct role for the bacteriophage λ FI gene product

Abstract A new in vitro bacteriophage λ DNA packaging system is described in which all the proteins necessary for head morphogenesis are supplied by extracts of plasmid-transformed cells. This assay is used to demonstrate that the λ FI gene product (gpFl) is necessary for maximal packaging efficiency when proheads and terminase are present in limiting amounts. A 100- to 200-fold decrease in packaging is seen when gpFI is omitted. gpFI is shown to act at and/or after the stage in packaging where proheads bind to the DNA:terminase complex.

Prediction of an ATP reactive center in the small subunit, gpNu1, of the phage lambda terminase enzyme

The small subunit of the bacteriophage lambda terminase enzyme, the product of the phages Nu1 gene, is shown to contain amino acid segments homologous to those present in a large number of ATPases. In keeping with these predictions, the purified protein has been found to hydrolyze ATP with a relatively low turnover number. Terminase holoenzyme is a known ATPase, and the biochemical significance of an ATP-interactive center situated in the gpNu1 subunit is discussed.

A sedimentation analysis of DNA found in Escherichia coli infected with phage λ mutants

Abstract A study was made of the phage DNA produced in Escherichia coli infected with mutants of λ unable to produce infectious particles because of defects in genes controlling head formation. Bacteria were irradiated with UV to eliminate host DNA synthesis and then infected with various λ sus mutants under conditions where subsequent radioactive labelling appeared exclusively in phage DNA. The intracellular DNA was extracted and examined by sedimentation in neutral and alkaline sucrose gradients. Mutants in genes A, B, C, D, and E cause blocks in DNA maturation which result in the accumulation of “rapidly sedimenting” species. Mutants in genes W and F accumulate much less of the rapidly sedimenting DNA and synthesize about 50–100% of the normal amounts of mature molecules as found in control infections. Intracellular phage DNA was purified from host material by density labelling and examined by electron microscopy. The “rapidly sedimenting” species appeared to be linear concatamers with lengths up to and exceeding four units. In an A mutant circular molecules were also found; these were both of monomer and multimer length (dimers and trimers). The proportion of immature DNA found by electron microscopy (long molecules) and by sedimentation (fast sedimenting DNA) agreed very well.

Probing the specificity of the S1 binding site of M222 mutants of subtilisin B. lentus with boronic acid inhibitors

Abstract Specificity differences between the S1-pockets of subtilisin B. lentus (SBL), and its M222C/S mutants have been explored with boronic acid inhibitors. Similar binding trends were noted, with 2,4-dichlorophenylboronic acid being the best overall inhibitor for each enzyme. In addition, a correlation between inhibitor binding and the electrophilicity of boron was found for both the M222C and M222S enzymes. Specificity differences between the S 1-pockets of subtilisin from B. lentus (SBL), and its M222C/S mutants, have been explored with boronic acid inhibitors. Similar binding trends were noted, with 2,4-dichlorophenyl boronic acid being the best overall inhibitor for each enzyme. In addition, a correlation between inhibitor binding and the electrophilicity of boron was found for both M222C and M222S enzymes.

Late stages in bacteriophage λ head morphogenesis: In vitro studies on the action of the bacteriophage λ D-gene and W-gene products

Abstract The in vitro maturation of bacteriophage λ can be divided into discrete steps. Concatemers of λ DNA bind terminase to form complex I. This DNA-terminase complex then binds a prohead to form a ternary complex (II). Complex II in turn can be converted to infectious phage by the addition of extracts containing the products of the phage genes D, W, FII , as well as phage tails. By using in vitro complementation assays gpD and gpW have been partially purified and their interactions with complex II studied. gpD can bind to complex II in vitro to form a new complex (III) which can be isolated by sedimentation on neutral sucrose gradients. This complex requires only the addition of gpW, gpFll, and phage tails to form mature phage particles. The sedimentation of complex III is virtually identical to that of complex II; however, the resistance of the former to inactivation by DNase is higher, likely due to the partial packaging of the DNA. In similar experiments it was shown that gpW cannot bind to complex II but can effectively interact with complex III. This latter reaction converts complex Ill to a DNase-resistant form which sediments in a manner identical to that of full phage heads (complex IV). After isolation of the complex IV only gpFll and tails are required for mature phage formation in vitro . gpW is a heat-stable protein of molecular weight approximately 10,000.

The Solution Structure of the Bacteriophage λ Head–Tail Joining Protein, gpFII

The bacteriophage lambda FII protein (gpFII) is a 117 residue structural protein found in the phage particle that is required for the joining of phage heads and tails at the last step of morphogenesis. We have performed biophysical experiments to show that gpFII is stable, monomeric, and reversibly folded. We have also determined the atomic resolution structure of gpFII using NMR spectroscopy. gpFII is shown to possess a novel fold consisting of seven beta-strands and a short alpha-helix. It also displays two large unstructured regions at the N terminus (residues 1-24) and in a large loop near the middle of the protein (residues 46-62). We speculate that these unstructured regions become structured when gpFII assembles into the phage particle, and that these conformational changes play an important role in regulating the assembly pathway. Alignment of the gpFII sequence with those of homologues from other lambdoid phages has allowed us to putatively identify distinct surfaces on the gpFII structure that mediate binding to the phage head and tail.

Altering the specificity of subtilisin B. Lentus by combining site-directed mutagenesis and chemical modification

The thiol side chain of the M222C mutant of the subtilisin from Bacillus lentus (SBL) has been chemically modified by methyl-, aminoethyl-, and sulfonatoethylthiosulfonate reagents. Introduction of charged residues into the active site of the enzyme reduced the catalytic efficiency with Suc-AAPF-pNA as the substrate, but resulted in better binding of sterically demanding boronic acid inhibitors.

DNA packaging in the lambdoid phages: The role of λ genes Nu1 and A

Abstract Of all the morphogenic genes of the coliphage λ, only Nu1 and A are both necessary and sufficient for terminase activity in vitro . Previously we have reported that extracts of Nu 1 − - and Aam − -infected cells will not complement each other to promote packaging in an in vitro assay (A. Becker, H. Murialdo, and M. Gold, Virology , 78 , 277–290, 1977). We report here that such complementation is possible under certain conditions; furthermore, complementation appears to proceed in a noncatalytic fashion. The product of gene Nu1 (gpNu1) therefore does not appear to be involved in the synthesis of the product of gene A (gpA). We discuss the hypothesis that λ terminase is a heterodimer consisting of gpNu1 and gpA subunits, and show that neither of these subunits alone is able to promote either the DNA-cutting or prohead-filling events of packaging.