Joel F. Schildbach

Assembly and mechanisms of bacterial type IV secretion machines.

Type IV secretion occurs across a wide range of prokaryotic cell envelopes: Gram-negative, Gram-positive, cell wall-less bacteria and some archaea. This diversity is reflected in the heterogeneity of components that constitute the secretion machines. Macromolecules are secreted in an ATP-dependent process using an envelope-spanning multi-protein channel. Similar to the type III systems, this apparatus extends beyond the cell surface as a pilus structure important for direct contact and penetration of the recipient cell surface. Type IV systems are remarkably versatile in that they mobilize a broad range of substrates, including single proteins, protein complexes, DNA and nucleoprotein complexes, across the cell envelope. These machines have broad clinical significance not only for delivering bacterial toxins or effector proteins directly into targeted host cells, but also for direct involvement in phenomena such as biofilm formation and the rapid horizontal spread of antibiotic resistance genes among the microbial community.

Structure and Specificity of the Anti-Digoxin Antibody 40-50

We determined the sequence, specificity for structurally related cardenolides, and three-dimensional structure of the anti-digoxin antibody 40-50 Fab in complex with ouabain. The 40-50 antibody does not share close sequence homology with other high-affinity anti-digoxin antibodies. Measurement of the binding constants of structurally distinct digoxin analogs indicated a well-defined specificity pattern also distinct from other anti-digoxin antibodies. The 40-50-ouabain Fab complex crystallizes in space group C2 with cell dimensions of a = 93.7 A, b = 84.8 A, c = 70.1 A, beta = 128.0 degrees. The structure of the complex was determined by X-ray crystallography and refined at a resolution of 2.7 A. The hapten is bound in a pocket extending as a groove from the center of the combining site across the light chain variable domain, with five of the six complementarity-determining regions involved in interactions with the hapten. Approximately three-quarters of the hapten surface area is buried in the complex; two hydrogen bonds are formed between the antibody and hapten. The surface area of the antibody combining site buried by ouabain is contributed equally by the light and heavy chain variable domains. Over half of the surface area buried on the Fab consists of the aromatic side-chains. The surface complementarity between hapten and antibody is sufficient to make the complex specific for only one lactone ring conformation in the hapten. The crystal structure of the 40-50-ouabain complex allows qualitative explanation of the observed fine specificities of 40-50, including that for the binding of haptens substituted at the 16 and 12 positions. Comparison of the crystal structures of 40-50 complexed with ouabain and the previously determined 26-10 anti-digoxin Fab complexed with digoxin, demonstrates that the antibodies bind these structurally related haptens in different orientations, consistent with their different fine specificities. These results demonstrate that the immune system can generate antibodies that provide diverse structural solutions to the binding of even small molecules.

Using Fluorophore-labeled Oligonucleotides to Measure Affinities of Protein-DNA Interactions

Changes in fluorescence emission intensity and anisotropy can reflect changes in the environment and molecular motion of a fluorophore. Researchers can capitalize on these characteristics to assess the affinity and specificity of DNA-binding proteins using fluorophore-labeled oligonucleotides. While there are many advantages to measuring binding using fluorescent oligonucleotides, there are also some distinct disadvantages. Here we describe some of the relevant issues for the novice, illustrating key points using data collected with a variety of labeled oligonucleotides and the relaxase domain of F plasmid TraI. Topics include selection of a fluorophore, experimental design using a fluorometer equipped with an automatic titrating unit, and analysis of direct binding and competition assays.

Subdomain organization and catalytic residues of the F factor TraI relaxase domain.

TraI from conjugative plasmid F factor is both a relaxase that sequence-specifically binds and cleaves single-stranded DNA (ssDNA) and a helicase that unwinds the plasmid during transfer. Using limited proteolysis of a TraI fragment, we generated a 36-kDa fragment (TraI36) retaining TraI ssDNA binding specificity and relaxase activity but lacking the ssDNA-dependent ATPase activity of the helicase. Further proteolytic digestion of TraI36 generates stable N-terminal 26-kDa (TraI26) and C-terminal 7-kDa fragments. Both TraI36 and TraI26 are stably folded and unfold in a highly cooperative manner, but TraI26 lacks affinity for ssDNA. Mutational analysis of TraI36 indicates that N-terminal residues Tyr(16) and Tyr(17) are required for efficient ssDNA cleavage but not for high-affinity ssDNA binding. Although the TraI36 N-terminus provides the relaxase catalytic residues, both N- and C-terminal structural domains participate in binding, suggesting that both domains combine to form the TraI relaxase active site.

Examination of an inverted repeat within the F factor origin of transfer: context dependence of F TraI relaxase DNA specificity

Prior to conjugative transfer of plasmids, one plasmid strand is cleaved in a site- and strand-specific manner by an enzyme called a relaxase or nickase. In F and related plasmids, an inverted repeat is located near the plasmid strand cleavage site, and others have proposed that the ability of this sequence to form a hairpin when in single-stranded form is important for transfer. Substitutions were introduced into a cloned F oriT region and their effects on plasmid transfer were assessed. For those substitutions that substantially reduced transfer, the results generally correlated with effects on in vitro binding of oligonucleotides to the F TraI relaxase domain rather than with predicted effects on hairpin formation. One substitution shown previously to dramatically reduce both plasmid transfer and in vitro binding to a 17-base oligonucleotide had little apparent effect on binding to a 30-base oligonucleotide that contained the hairpin region. Results from subsequent experiments strongly suggest that the relaxase domain can bind to hairpin oligonucleotides in two distinct manners with different sequence specificities, and that the protein binds the oligonucleotides at the same or overlapping sites.

Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

Conjugative plasmid transfer is an important mechanism for diversifying prokaryotic genomes and disseminating antibiotic resistance. Relaxases are conjugative plasmid-encoded proteins essential for plasmid transfer. Relaxases bind and cleave one plasmid strand site- and sequence-specifically before transfer of the cleaved strand. TraI36, a domain of F plasmid TraI that contains relaxase activity, binds a plasmid sequence in single-stranded form with subnanomolar KD and high sequence specificity. Despite 91% amino acid sequence identity, TraI36 domains from plasmids F and R100 discriminate between binding sites. The binding sites differ by 2 of 11 bases, but both proteins bind their cognate site with three orders of magnitude higher affinity than the other site. To identify specificity determinants, we generated variants having R100 amino acids in the F TraI36 background. Although most retain F specificity, the Q193R/R201Q variant binds the R100 site with 10-fold greater affinity than the F site. The reverse switch (R193Q/Q201R) in R100 TraI36 confers a wild-type F specificity on the variant. Nonadditivity of individual amino acid and base contributions to recognition suggests that the specificity difference derives from multiple interactions. The F TraI36 crystal structure shows positions 193 and 201 form opposite sides of a pocket within the binding cleft, suggesting binding involves knob-into-hole interactions. Specificity is presumably modulated by altering the composition of the pocket. Our results demonstrate that F-like relaxases can switch between highly sequence-specific recognition of different sequences with minimal amino acid substitution.

Tracking F plasmid TraI relaxase processing reactions provides insight into F plasmid transfer

Early in F plasmid conjugative transfer, the F relaxase, TraI, cleaves one plasmid strand at a site within the origin of transfer called nic. The reaction covalently links TraI Tyr16 to the 5′-ssDNA phosphate. Ultimately, TraI reverses the cleavage reaction to circularize the plasmid strand. The joining reaction requires a ssDNA 3′-hydroxyl; a second cleavage reaction at nic, regenerated by extension from the plasmid cleavage site, may generate this hydroxyl. Here we confirm that TraI is transported to the recipient during transfer. We track the secondary cleavage reaction and provide evidence it occurs in the donor and F ssDNA is transferred to the recipient with a free 3′-hydroxyl. Phe substitutions for four Tyr within the TraI active site implicate only Tyr16 in the two cleavage reactions required for transfer. Therefore, two TraI molecules are required for F plasmid transfer. Analysis of TraI translocation on various linear and circular ssDNA substrates supports the assertion that TraI slowly dissociates from the 3′-end of cleaved F plasmid, likely a characteristic essential for plasmid re-circularization.

Roles of Active Site Residues and the HUH Motif of the F Plasmid TraI Relaxase

Bacterial conjugation, transfer of a single strand of a conjugative plasmid between bacteria, requires sequence-specific single-stranded DNA endonucleases called relaxases or nickases. Relaxases contain an HUH (His-hydrophobe-His) motif, part of a three-His cluster that binds a divalent cation required for the cleavage reaction. Crystal structures of the F plasmid TraI relaxase domain, with and without bound single-stranded DNA, revealed an extensive network of interactions involving HUH and other residues. Here we study the roles of these residues in TraI function. Whereas substitutions for the three His residues alter metal-binding properties of the protein, the same substitution at each position elicits different effects, indicating that the residues contribute asymmetrically to metal binding. Substitutions for a conserved Asp that interacts with one HUH His demonstrate that the Asp modulates metal affinity despite its distance from the metal. The bound metal enhances binding of ssDNA to the protein, consistent with a role for the metal in positioning the scissile phosphate for cleavage. Most substitutions tested caused significantly reduced in vitro cleavage activities and in vivo transfer efficiencies. In summary, the results suggest that the metal-binding His cluster in TraI is a finely tuned structure that achieves a sufficient affinity for metal while avoiding the unfavorable electrostatics that would result from placing an acidic residue near the scissile phosphate of the bound ssDNA.

Single-Stranded DNA Binding by F TraI Relaxase and Helicase Domains Is Coordinately Regulated

Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal approximately 300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188:6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer.

An intrastrand three-DNA-base interaction is a key specificity determinant of F transfer initiation and of F TraI relaxase DNA recognition and cleavage

Bacterial conjugation, transfer of a single conjugative plasmid strand between bacteria, diversifies prokaryotic genomes and disseminates antibiotic resistance genes. As a prerequisite for transfer, plasmid-encoded relaxases bind to and cleave the transferred plasmid strand with sequence specificity. The crystal structure of the F TraI relaxase domain with bound single-stranded DNA suggests binding specificity is partly determined by an intrastrand three-way base-pairing interaction. We showed previously that single substitutions for the three interacting bases could significantly reduce binding. Here we examine the effect of single and double base substitutions at these positions on plasmid mobilization. Many substitutions reduce transfer, although the detrimental effects of some substitutions can be partially overcome by substitutions at a second site. We measured the affinity of the F TraI relaxase domain for several DNA sequence variants. While reduced transfer generally correlates with reduced binding affinity, some oriT variants transfer with an efficiency different than expected from their binding affinities, indicating ssDNA binding and cleavage do not correlate absolutely. Oligonucleotide cleavage assay results suggest the essential function of the three-base interaction may be to position the scissile phosphate for cleavage, rather than to directly contribute to binding affinity.