The phage lambda terminase enzyme: 1. Reconstitution of the holoenzyme from the individual subunits enhances the thermal stability of the small subunit.

The terminase enzyme from bacteriophage lambda is a hetero-trimeric complex composed of the viral gpA and gpNu1 proteins (gpA1.gpNu1(2)) and is responsible for packaging a single genome within the viral capsid. Current expression systems for these proteins require thermal induction which may be responsible for the formation of insoluble aggregates observed in E. coli. We report the re-cloning of the terminase subunits into vectors which allow low temperature induction. While this has resulted in increased solubility of the large gpA subunit of the enzyme, the small gpNu1 subunit remains insoluble under all conditions examined. This paper describes the solublization of gpNu1 with guanidinium hydrochloride and purification of the protein to homogeneity. Reconstitution of the enzyme from the individually purified subunits yields a catalytically-competent complex which exhibits activity identical to wild-type enzyme. Thermal denaturation of the proteins was monitored by circular dichroism (CD) spectroscopy and demonstrates that while unfolding of gpA is irreversible, the gpNu1 subunit refolds into a conformation which is essentially identical to the pre-heated protein. Moreover, while denaturation of gpA is highly cooperative, the small subunit unfolds over a wide temperature range and with thermodynamic parameters lower than expected for a small globular protein. Thermally-induced denaturation of the enzyme reconstituted from the individual subunits is highly cooperative with no evidence of multiple transitions. Our data demonstrate that the terminase subunits directly interact in solution, and that this interaction alters the thermal stability of the smaller gpNu1 subunit. The implication of these results with respect to assembly of a catalytically competent enzyme complex are discussed.

The phage lambda terminase enzyme: 2. Refolding of the gpNu1 subunit from the detergent-denatured and guanidinium hydrochloride-denatured state yields different oligomerization states and altered protein stabilities.

The terminase enzyme from bacteriophage lambda is responsible for packaging a single genome within the viral capsid. Gold and co-workers have developed a scheme for the solubilization of the small terminase subunit (gpNu1) from inclusion bodies using the strong detergent sarkosyl and purification of the protein to homogeneity (gpNu1SRK) (Parris et al., J Biol Chem 1994;269:13564-13574). We have developed a similar purification scheme except that guanidinium hydrochloride was used to denature the insoluble protein (gpNu1GDN). The circular dichroism (CD) spectra of both protein preparations suggest that they are predominantly alpha-helical when purified and stored in Tris buffers. Moreover, thermal denaturation of the proteins thus purified yielded similar thermodynamic parameters for unfolding (T(m), delta Hm and delta Sm of unfolding of approximately 306 K, approximately 22 kcal/mol and approximately 70 cal/mol.K, respectively). Interestingly, however, when the proteins were purified and stored in imidazole buffers, the gpNu1SRK preparation lost a significant amount of secondary structure and was more stable to both thermally-induced and guanidinium HCl-induced denaturation than was gpNu1GDN. The purified gpNu1 monomers oligomerize into apparent tetramers and hexamers in solution and the distribution between these two oligomeric states and into higher order aggregates depends upon buffer composition, salt concentration and protein concentration. Moreover, differences in the oligomerization state of gpNu1SRK and gpNu1GDN under identical buffer conditions were observed. The significance of these results with respect to the biological role of the phage lambda gpNu1 protein are discussed.

Assembly of a nucleoprotein complex required for DNA packaging by bacteriophage lambda.

A critical step in the assembly of bacteriophage lambda is the excision of a single genome from a concatemeric DNA precursor and insertion of genomic DNA into an empty viral capsid. DNA packaging is mediated by the lambda proteins gpNu1 and gpA, which form an enzyme complex known as terminase. Initiation of the packaging process requires assembly of the terminase subunits onto cos, the lambda DNA packaging sequence, and nicking of the duplex, thus forming the 12-base-pair "sticky" ends of the mature genome. We have utilized gel-retardation techniques to examine the interaction of gpNu1, gpA, and terminase holoenzyme with DNA. Our data demonstrate that gpNu1 interacts specifically with cos-containing DNA, forming three gel-retarded complexes. Similarly, the larger gpA subunit binds to DNA, forming two complexes; however, this subunit forms similar complexes with DNA substrates of random sequence. All of the nucleoprotein complexes examined are disrupted by elevated concentrations of NaCl and we suggest that altered DNA binding is responsible for the extreme salt sensitivity of the endonuclease activity of the enzyme [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065]. DNA binding by each subunit is strongly affected by the presence of the other, with 10- and 3-fold increases in the affinity of gpNu1 and gpA, respectively, for DNA. Moreover, our data suggest that the terminase subunits interact in solution prior to DNA binding. Finally, we provide evidence that complex I, the first stable intermediate in the packaging pathway, is composed of the mature left genome end bound to the terminase subunits and demonstrate that dissociation of the complex is quite slow (t1/2 > 8 h). The significance of these data with respect to terminase-mediated genome packaging is discussed.

Identification of class II major histocompatibility complex and T cell receptor binding sites in the superantigen toxic shock syndrome toxin 1.

Superantigens, in association with class II major histocompatibility complex (MHC) molecules, activate T cells bearing particular beta chain variable domains of the T cell receptor (TCR). Unlike conventional peptide antigens, superantigens bind as intact proteins to TCR and MHC molecules outside their peptide binding sites. To characterize these interactions at the molecular level, random point mutations were generated in the gene encoding toxic shock syndrome toxin 1, a bacterial superantigen associated with toxic shock syndrome. Functionally impaired mutants were identified based on their lack of murine and human T cell stimulatory activities, and experiments analyzing binding to human histocompatibility leukocyte antigen-DR molecules differentiated residues involved in MHC from TCR binding. The results showed that the great majority of mutations are clustered in two distinct regions of the toxic shock syndrome toxin 1 molecule. The class II MHC binding site is located in the hydrophobic region of the NH2-terminal domain, and the TCR binding site is primarily in the major central groove of the COOH-terminal domain. These studies provide insight into the interactions necessary for superantigen-mediated disease in humans.