The three-dimensional structure of a Tn5 transposase-related protein determined to 2.9-A resolution.

Transposon Tn5 employs a unique means of self-regulation by expressing a truncated version of the transposase enzyme that acts as an inhibitor. The inhibitor protein differs from the full-length transposase only by the absence of the first 55 N-terminal amino acid residues. It contains the catalytic active site of transposase and a C-terminal domain involved in protein-protein interactions. The three-dimensional structure of Tn5 inhibitor determined to 2.9-A resolution is reported here. A portion of the protein fold of the catalytic core domain is similar to the folds of human immunodeficiency virus-1 integrase, avian sarcoma virus integrase, and bacteriophage Mu transposase. The Tn5 inhibitor contains an insertion that extends the beta-sheet of the catalytic core from 5 to 9 strands. All three of the conserved residues that make up the "DDE" motif of the active site are visible in the structure. An arginine residue that is strictly conserved among the IS4 family of bacterial transposases is present at the center of the active site, suggesting a catalytic motif of "DDRE." A novel C-terminal domain forms a dimer interface across a crystallographic 2-fold axis. Although this dimer represents the structure of the inhibited complex, it provides insight into the structure of the synaptic complex.

A mechanism for Tn5 inhibition. carboxyl-terminal dimerization.

Tn5 is unique among prokaryotic transposable elements in that it encodes a special inhibitor protein identical to the Tn5 transposase except lacking a short NH2-terminal DNA binding sequence. This protein regulates transposition through nonproductive protein-protein interactions with transposase. We have studied the mechanism of Tn5 inhibition in vitro and find that a heterodimeric complex between the inhibitor and transposase is critical for inhibition, probably via a DNA-bound form of transposase. Two dimerization domains are known in the inhibitor/transposase shared sequence, and we show that the COOH-terminal domain is necessary for inhibition, correlating with the ability of the inhibitor protein to homodimerize via this domain. This regulatory complex may provide clues to the structures of functional synaptic complexes. Additionally, we find that NH2- and COOH-terminal regions of transposase or inhibitor are in functional contact. The NH2 terminus appears to occlude transposase homodimerization (hypothetically mediated by the COOH terminus), an effect that might contribute to productive transposition. Conversely, a deletion of the COOH terminus uncovers a secondary DNA binding region in the inhibitor protein which is probably located near the NH2 terminus.

Functional characterization of the Tn5 transposase by limited proteolysis.

The 476 amino acid Tn5 transposase catalyzes DNA cutting and joining reactions that cleave the Tn5 transposon from donor DNA and integrate it into a target site. Protein-DNA and protein-protein interactions are important for this tranposition process. A truncated transposase variant, the inhibitor, decreases transposition rates via the formation of nonproductive complexes with transposase. Here, the inhibitor and the transposase are shown to have similar secondary and tertiary folding. Using limited proteolysis, the transposase has been examined structurally and functionally. A DNA binding region was localized to the N-terminal 113 amino acids. Generally, the N terminus of transposase is sensitive to proteolysis but can be protected by DNA. Two regions are predicted to contain determinants for protein-protein interactions, encompassing residues 114-314 and 441-476. The dimerization regions appear to be distinct and may have separate functions, one involved in synaptic complex formation and one involved in nonproductive multimerization. Furthermore, predicted catalytic regions are shown to lie between major areas of proteolysis.

A functional analysis of the Tn5 transposase. Identification of domains required for DNA binding and multimerization.

A series of deletions were constructed in the 476 amino acid Tn5 transposase in order to assemble an initial domain structure for this protein. The first four amino acids were found to be important for transposition activity but not for DNA binding to the Tn5 outside end (OE). Larger amino-terminal deletions result in the complete loss of transposition in vivo and the concomitant loss of specific DNA binding. Four point mutants and a six base-pair deletion in the amino terminus between residues 20 and 36 were also found to impair DNA binding to the OE. Analysis of a series of carboxy-terminal deletions has revealed that the carboxy terminus may actually mask the DNA binding domain, since deletions to residues 388 and 370 result in a large increase in DNA binding activity. In addition, the carboxy-terminal deletion to residue 370 results in a significant increase in the mobility of the Tnp-OE complex indicative of a change in the oligomeric state of this complex. Further carboxy-terminal deletions beyond residue 370 also abolished DNA binding activity. These results indicate that the first four amino acids of Tnp are important for transposition but not DNA binding, a region between residues 5 and 36 is critical for DNA binding, the wild-type carboxy terminus acts to inhibit DNA binding, and that a region towards the carboxy terminus, defined by residues 370 to 387, is critical for Tnp multimeric interactions.