The Three Dimensional Structure of the Lysozyme from Bacteriophage T4

Abstract

The three dimensional structure of the lysozyme from bacteriophage T4 has been determined from a 2.5 A resolution electron density map. About 60% of the molecule is in a helical conformation and there is one region consisting of antiparallel (-structure. The polypeptide backbone folds into two' distinct lobes linked in part by a long helix. In the region between the two lobes, there is a cleft which deepens into a hole or cavity, about 6-8 A in diameter, extending'from one side of the molecule to the other. This opening is closed off by side chains which extend to within 3-5 A of each other. A number of mutant lysozymes in which residues in the vicinity of the opening are modified have markedly reduced catalytic activity, suggesting that this region of the molecule may be catalytically important. The three dimensional structure of -T4 phage lysozyme is quite different from that of hen egg-white lysozyme although it is not clear at this time whether or not the mechanisms of catalysis of the respective enzymes are related.T4 phage lysozyme is an enzyme produced in cells of Escherichia coli after infection with bacteriophage T4. The enzyme has similar catalytic activity to that of hen egg-white lysozyme, both being endoacetylmuramidases (1). The molecular weight of the enzyme is 18,700, and the amino-acid sequence has been determined (2). Furthermore, a number of lysozymes have been isolated from mutant strains carrying frame-shift or amber mutations in the phage genome and have been used to demonstrate in vivo certain features of the genetic code (3-5).In this preliminary report, we describe the three dimensional structure of T4 phage lysozyme as determined by x-ray crystallography from a 2.5 A resolution electron density map. Additional details will be given in a subsequent publication. CrystallizationThe protein was purified using essentially the method of Tsugita et al.(1) except that 1 mM mercaptoethanol was added to all buffers. Conditions for obtaining the crystals have been described previously (6). Before x-ray photography the crystals were equilibrated with a standard mother liquor consisting of 1.05 M K2HPO4, 1.26 M NaH2PO4, 0.23 M NaCl, 1.4 mM mercaptoethanol, pH 6.7. In this solution the crystals float.The crystals have space group P3221 with cell dimensions a = b = 61.1 A, c = 96.3 A and one molecule in each of the six asymmetric units. Data collectionDiffraction data were recorded photographically using conventional Buerger precession cameras and integrated intensities measured with a computer controlled drum film scanner (7). Sets of 12 films sufficed to measure 92% of the data to 2.5 A and 53% of the data between 2.4 A and 2.5 A. With the films used, the Bijvoet differences were obtained for about half the reflections. Systematic errors in the measurement of the Friedel pairs were reduced by a method of local scaling to be described elsewhere (ref 8, B. W. Matthews et al., manuscript in preparation). Altogether, about 27,000 intensities were measured for the parent crystals and the two heav...

Lessons from the lysozyme of phage T4

“…This was one of the most striking features of the original structure determination and it was remarked at the time that the enzyme would presumably have to undergo a fairly large conformational change to admit its peptidoglycan substrate. 61 Consistent with this, there are many mutant crystal structures, which display large variations in the hinge-bending angle between the N-and C-terminal domain.…”

A backbone-based theory of protein folding

“…The scaffold is robust to changes in the protein's amino acid sequence. For example, there are Ϸ130 structures of phage lysozyme mutants in the protein databank (100); all have conformations that closely resemble the parent structure (101). Presumably then, the molten globule also is robust to changes in sequence.…”

Tess: A geometric hashing algorithm for deriving 3D coordinate templates for searching structural databases. Application to enzyme active sites

“…The catalytic residues in horse lysozyme (Tsuge et al, 1992) have an RMSD of 2.52 8, from the consensus Prokaryotic: Bacteriophage T4 lysozyme T4 lysozyme is produced late in the infection of Escherichia coli by T4 bacteriophage. The structure was first determined by Matthews ind Remington (1974). This enzyme has been used as a model to study the effects of mutations on protein stability and function and there are 160 different mutant forms deposited in the PDB (e.g., Alber et al, 1987;Weaver & Matthews, 1987).…”