References:

Silverman, M., and M. Simon, in Mobile Genetic Elements, J.A. Shapiro, ed.. Academic Press, 1983.

Hin recombinase catalyzes a site-specific DNA inversion in the Salmonella chromosome, shown schematically at left.

(A) The recombination event regulates the alternate expression of two flagellin genes, H2 and H1 (not shown). The hin gene encodes hin recombinase protein and lies between two 14 bp inverted repeats, hixL and hixR. Also contained between these repeats is the H2 promoter (P), responsible for driving transcription of the H2 operon. The operon is transcribed when the promoter is oriented near the structural genes H2 and rH1. This transcription is followed by translation of the H2 flagellin protein and of the rH1 protein, a repressor of H1 flagellin gene transcription. Thus, in the situation just described, the H2 flagellin gene is expressed but the H1 flagellin gene is silent.

(B) Hin recombinase is responsible for binding hixL and hixR, looping out intervening DNA. There is also a cis-acting site on the DNA that is bound by two dimers of the Fis protein (not shown), forming a synaptic complex with hixL, hixR, and the bound hin protein. This complex is called the invertasome complex, and its formation permits the recombination sites to be properly aligned for the recombination event. Hin recombinase can then catalyze homologous recombination between the repeats, now oriented in parallel. This results in a reversible switch of the orientation of the ~1,000 bp segment containing the hin gene and the H2 promoter (P).

(C) As a consequence of the inversion, transcription of the H2 operon is shut off, and the resulting absence of rH1 repressor allows expression of the H1 gene, producing H1 flagellin. 

Shown at left is the carboxy-terminal 52-amino acid DNA-binding domain (the Hin domain) of the Hin recombinase molecule, complexed with a hixL DNA recombination half site. The Hin domain contains a three alpha helix bundle <> with the carboxy-terminal alpha helix (helix 3) of this bundle inserted into the major goove of the DNA parallel to the base pairs (not to the floor of the groove itself). Alpha helix 1 is oriented parallel to the axis of the DNA, and alpha helix 2 is positioned in a almost antiparallel manner to helix 1. There is an angle of -25^o between the helix axes. Helices 2 and 3 form a helix-turn-helix motif (HTH) that is similar to those found in other prokaryotic regulatory DNA-binding proteins.

The three alpha helices are amphipathic, having hydrophobic residues tightly packed agaist one another in a hydrophobic core <>. Ile¹⁵² and Leu¹⁵⁶ of helix 1 interact with Leu¹⁶⁵ and Phe¹⁶⁹ of helix 2, and Val¹⁷³, Leu¹⁷⁶, and Phe¹⁸⁰ of helix 3 also point into this hydophobic core. Ile¹⁴⁴, located on the amino-terminal arm of the Hin protein, closes this hydrophobic pocket <>. These hydrophobic forces play a major role in the stabilization of the folding of the Hin protein.

There are also hydrogen bonds in the Hin domain that supplement the stabiliztion of the peptide by the hydrophobic interactions. For example, Arg¹⁶², located at the beginning of helix 2, is hydrogen bonded to the main chain carbonyl oxygens of Phe¹⁸⁰, which is the final residue of helix 3, and Pro¹⁸¹ <> . The Hin peptide is further stabilized by the orientation of most of the charged side chains in the Hin domain. These are either in contact with the DNA, or exposed to solvent (water) <>.

The Hin domain also includes two flanking extended amino- and carboxy-terminal polypeptides that contact the bases of the DNA along two different regions in the minor groove <>. 

Hin recombinase binds to each recombination site on standard B-form DNA as a dimer, and the final 52 amino acids of the two monomers bind to a 26-bp recombination site. The recombination site is made up of two 12-bp inverted repeats seperated by a 2-bp core region where DNA strand exchange occurs (13 of these base pairs are shown here) <> . The amino-terminal catalytic domain of Hin recombinase, consisting of 138 amino acids, is positioned in part over the core nucleotides (not shown).

When complexed with Hin recombinase, the DNA remains relatively straight and is not significantly bent around the protein. This could be explained in part by the fact that the DNA half site contains a short run of five AT base pairs <> . This segment of DNA may be regarded as a region of A-tract DNA, characterized by a straight, unbent axis, a large propeller twist, and a narrow minor groove. However, when this small section of A-tract DNA is complexed with the Hin protein, the minor groove is considerably wider (approx. 6.5-8.5 Å) than it would be for typical A-tracts (3.5-4.5 Å). Also, propeller twist is large (approximately -16^o) all along the DNA-Hin complex, but is not significantly larger in the A-tract region.

The Hin protein contacts an unusually large amount of surface area on the DNA <> : the DNA half-site monomer loses 1816 Ų of its static solvent accessible surface area when it is bound by the Hin protein. 

A. Overview

As shown above, the carboxy-terminal alpha helix of the Hin protein interacts with the major groove of the DNA, while the flanking amino- and carboxy-terminal chains <> interact with the minor groove. Specific binding of the Hin peptide to DNA requires both the major groove interactions involving alpha helix 3 and minor groove interactions involving the amino-terminal sequence Gly¹³⁹-Arg¹⁴⁰-Pro¹⁴¹-Arg¹⁴²<>. The carboxy-terminal eight-amino acids also contribute to base sequence recognition, and cross the phosphodiester backbone of DNA, inserting into the minor groove in a novel DNA-protein complex <>. The binding affinity of the Hin dimer to the full recombination site is approximately 100-fold higher than the binding affinity of the Hin monomer to a recombination half-site, indicating that cooperative interactions between the Hin monomers may contribute to sequence recognition as well.

B. Major Groove Interactions

1. Nonspecific interactions

Helix 3 is the only helix in the Hin protein that interacts directly with DNA (the other two helices are not positioned close enough to the DNA to allow any interaction to take place). However, Gln¹⁶³, at the amino terminus of helix 2, indirectly contacts the DNA through a hydrogen bond to Tyr¹⁷⁷ in helix 3. Tyr¹⁷⁷ in turn interacts with phosphate P19 on the DNA <> . There are five nonspecific interactions between helix 3 and the phosphate backbone that help to position this helix properly in the major groove and allow for specific recognition interactions. The side chain of Tyr¹⁷⁷ interacts with phosphate P19 on one edge of the major groove, while Tyr¹⁷⁹ interacts with phosphate P8 directly across the groove on its other edge <> . Additionally, one of the terminal -NH₂ groups of the Arg¹⁷⁸ side chain forms a hydrogen bond with the remaining oxygen of phosphate P8 <> . Also, the side chain of Thr¹⁷⁵ and the main chain amide of Gly¹⁷² form hydrogen bonds with phosphate P9 <> .

2. Specific Interactions

Specific base sequence recognition between the Hin peptide and DNA also occurs, involving only the side chains of Ser¹⁷⁴ and Arg¹⁷⁸, and two water molecules (not shown) <>. The side chain of Ser¹⁷⁴ forms a hydrogen bond with the N-7 atom of base A10, and one alpha helix-turn away from this position, the terminal -NH₂ of Arg¹⁷⁸ forms a hydrogen bond with the N-7 atom of base G9. Another nitrogen <> of Arg¹⁷⁸ donates a hydrogen bond to water molecule 1, which hydrogen bonds to the O-4 atom of base T22. One of the remaining protons of water molecule 1 forms a hydrogen bond with water molecule 2, which also forms hydrogen bonds with the N-6 and N-7 atoms of base A21 and with the carbonyl oxygen of Ser¹⁷⁴. The interaction of Hin with DNA through these solvent H₂O molecules allows Hin to "read" adjacent AT's in the major groove.

On the basis of these specific and nonspecific interactions, it is possible to imagine a mechanism whereby Hin could slide along the DNA in a nonspecific fashion until it encoutered its correct recognition sequence.

C. Minor Groove Interactions

1. The amino-terminal arm

<> The amino-terminal arm (Gly¹³⁹ to His¹⁴⁷) of the Hin peptide adopts an extended conformation, and Gly¹³⁹ and Arg¹⁴⁰ are located within the minor groove when the Hin peptide is bound to DNA. The side chain of Arg¹⁴⁰ forms a hydrogen bond with the N-3 atom of base A26, and the unusually high propeller twist (26^o) of this base pair allows for another hydrogen bond to be formed between the main chain amide of Arg¹⁴⁰ and the O-2 atom of base T6. Also, Gly¹³⁹ participates in Van der Waals interactions with base pair 5 <>.

Pro¹⁴¹ arches across one wall of the minor groove, and there is a hydrogen bond between Arg¹⁴² and phosphate P8 <>. This interaction may be involved in directing the amino-terminal arm of the Hin peptide into the minor groove. It is also be possible that Ile¹⁴⁴ (discussed above) is important in restricting the movement of the amino terminal arm, thereby positioning Arg¹⁴² favorably for hydrogen bonding to phosphate P8 <>.

2. The carboxyl-terminal tail

<> The carboxyl-terminal tail crosses the phosphodiester backbone of the DNA at the outer edge of the recombination site and then curves around to follow the minor groove back toward the center of the 13-bp recombination half-site. The six most caboxyl-terminal amino acid residues adopt an extended conformation and lie within the minor groove. However, the side chains of these amino acids make no contacts with the floor of the minor groove and instead point outward, leaving the polypeptide backbone to rest against the bases.

<> The main chain carbonyl group of Ile¹⁸⁵ forms a hydrogen bond with the N-2 atom of base G-14, the main chain -NH group of Lys¹⁸⁷ forms a hydrogen bond with to the O-2 atom of base T20, the main chain amide of Asn¹⁹⁰ interacts with the O-2 atom of base T22, and the side chain of Asn¹⁹⁰ interacts with the N-3 atom of base A10. 

The R.E.D. Gallery: Another image and brief description of Hin Recombinase (as well as several other molecules, including the Fis protein)

Current Research on the interaction of Hin recombinase with the Fis protein, and other levels regulation of transcription of the flagellin gene (Kelly T. Hughes)

Phase variation of Salmonella flagellar antigens

References:

Silverman, M., and M. Simon, in Mobile Genetic Elements, J.A. Shapiro, ed.. Academic Press, 1983.