Biomolecules at Kenyon XX HHMI at Kenyon xx Jmol Home xx Biology Dept xx COMMENTS and CORRECTIONS

Integration Host Factor

Josh Weber '11 and Phan Truong '11


I. Introduction

Integration host factor (IHF) was first discovered as a host factor for bacteriophage λ integration. IHF was found to assist in other processes that include high order protein-complexes such as in replication where it binds to oriC, transcriptional regulation where it can help initiate transcription by interacting with RNA polymerase, and site-specific recombination (Goosen and van de Putte, 1995). IHF’s primary function appears to be architectural. It binds to DNA in a sequence specific manner and introduces a sharp bend (>160°) in the DNA that facilitates interaction between components in nucleoprotein array.

IHF is member of minor groove-intercalating DNA-bending proteins. It is a small heterodimer with subunits that are approximately 30% identical in sequence. The structure of IHF is closely related to HU , a nonspecific DNA binding protein. The members of the IHF/HU family have been found in a broad range of prokaryotes (Oberto and Rouviere-Yaniv, 1996).

II. General Structure

The fold of IHF is essentially the same as that of HU . The subunits of IHF, IHFα and IHFβ, are intertwined to form a body with two long β sheet arms that extend from it . The arms interact exclusively with the minor groove of DNA and wrap around it. Each IHF subunit contains 5 Beta-sheets(S) and 3 Alpha-Helices(H) .The order of S and H is H1-H2-S1-S2-S'2-S'3-S3-H3 . The majority of the bending occurs at two kinks 9 base pairs(bp) apart and proline residues at the tip of the arm intercalates between base pairs .

The phosphate backbone contacts 26 positively charged side chains and interacts with the N-termini of all six helices of the heterodimer. The ends of H1 and H3 form a clamp by binding to opposite sides of the minor groove with respect to the intercalating proline. H2 forms a hydrogen bond from the bottom of the protein to adjacent DNA fragments.

III. Interaction of the Beta-arms and Proline

The proline residue in each arm is conserved in every member of the HU/IHF families. Proline is favored because its width allows extensive hydrophobic contact with the DNA bases , and its stubbiness allows formation of a hydrogen bond between the peptide backbone of the arm and N3 of A immediately 5’ to each kink . It could also stabilize the structure of the turn at the end of each arm. The DNA within half a turn in either direction of the kinks adopts a B-form structure with an unusually narrow minor groove.

IV. Clamping of the DNA

The backbone on each side of the body of IHF forms a tripartite clamp that binds across the minor groove. At the center of this is a turn between S1 and S2 that lie between two phosphates on opposite sides of the minor groove and is hydrogen bonded to both strands by successive amide nitrogens . The phosphates also interact with the N-termini of helices 1 and 3. On the right side, the clamp straddles the second portion of the consensus sequence and R46β extends from the S1-S2 turn, contacting edges of this conserved sequence. On the left side, the clamp straddles the minor groove of the A tract but doesn’t contact the bases.

V. Recognition of A-Tract Regions

The A tract is straight, has a narrow minor groove compared to a normal B-form minor groove , and high propeller twists between bases. It is recognized by its structure, not by sequence, and is preferred by IHF because the narrow minor groove fits into the clamp better. In this tract and the kinks, is a spine of hydration, a well-ordered string of water molecules. The minor groove faces the protein and shields the spine from the solvent, leaving unbounded hydrogen bond donors and acceptors. Side chain S47α, from the S1-S2 turn, points into the minor groove and provides hydrogen bond donors and acceptors . At the top of the body, three β strands of each subunit are bridged by a row of water molecules and the narrow minor groove lies parallel to these strands. A thin sheet of water extends from the bridging subunits to the DNA’s spine of hydration. The second consensus sequence is in the exceptionally narrow minor groove and R46β replaces the spine of hydration.

VI. Recognition of Consensus Sequence

There are only two conserved sequences in the IHF binding site [concensus sequence] . The first sequence is 5'-TATCAA-3' and is contacted by the arm of IHFα. R42β and R46β reach into the minor groove of the second concensus sequence, 5'-TTG-3',and hydrogen bond to the conserved bases. These contacts may help to select the unusual DNA conformation that favors particular bases at these positions. Base pairs 36 and 37 are the only bases that are conserved in every binding site. These pairs are highly buckled because of the intercalation of proline between bp 37 and 38. This buckling resolves at bp 35 and creates a tilt of 32° between T35 and C36. The conservation of A37 might be because of a need to satisfy a highly distorted DNA structure and a limited number of protein contacts. The large roll angle between bp 37 and 38 allows an interaction between methyl groups in the minor groove: T37 is inserted into a hydrophobic pocket between the ribose moiety and the methyl of T38. The arm of IHFα is inserted into the minor groove more deeply than the arm of IHFβ and to the lack of sequence specificity in this region.

The second consensus sequence is recognized by the body of IHF. R46β extends from the S1-S2 turn into minor groove and makes direct and indirect water mediated hydrogen bonds to the three conserved bases. Its guanidinium is in the center of the groove and is enforced by a chain of salt bridges. Both sides of the carboxylate of E44β form salt bridges , one to the back of R46β and the other to R42β. R42β makes bridges to the phosphate of A41. E44β acts as a buttress holding R46β in place. The narrowness of the minor groove allows the aliphatic portion of R46β to make van der Waals ineractions with ribose moieties of the DNA backbone on both sides of the grooves.

VII. Implication

IHF contributes to the biological functions of many prokaryotes. One example is its role in the excision of λ prophage. IHF bends the DNA, allowing the λ integrase to bind to its promoter and contact the cleavage site. It has also been suggested that IHF can assist RNA polymerase to initiate transcription by presenting a portion of the promoter sequence to the relevant portion of RNA polymerase. Since there is a large region of the major groove that is left open for more interactions, other proteins may interact with these sites. When IHF narrows the minor groove, it does so by widening the major groove. When this occurs, these sites may adopt a better conformation for other proteins to bind.

In order to bend DNA, proteins must overcome the forces that usually keep the DNA straight. Some of the most important of the forces that must be overcome are the repulsion of the phosphate backbone charges and the energy from base stacking. By placing a large positive surface on the inside of the DNA bend, IHF is able to asymmetrically neutralize the double helix. To counteract the energy of base stacking, IHF intercalates a hydrophobic side chain, the proline at the tip of the β arms, between the base pairs.

The sequence specificity of the binding of IHF to DNA is influenced by the sequence-dependent conformability of DNA, shown by the amount of direct protein-DNA contacts made. Since the minor groove has less unique features than the major groove, this theme could be found in many minor groove binding proteins.

The conformation of the A tract that is observed in the crystallization of the IHF-DNA complex is also observed in the crystal structure of naked duplex DNA. The features of the A tract are used to generate optimal contacts between IHF and the helix. This provides evidence for the significance of structure of the A tract as a recognition element for other proteins that interact with A-rich sequences.

HU is 40% identical in sequence to IHF but binds DNA nonspecifically, even though main features such as the proline residue at the tip of each arm, and the distribution of positive charge are conserved in each protein. The sequence specificity of IHF must arise from the small differences between it and HU. In HU, the R46 of IHFβ that contacts the conserved bases is replaced with either valine or isoleucine. Structural differences in this region also play a role in the sequence specificity of IHF. Helix 1 of IHFα is displaced axially by 1.3 Å toward its C-terminus, compared to HU and even IHFβ. This allows the phosphate nearest its N-terminus to be tucked into the protein deeper, resulting in an odd variation in twist at this position. The difference in sequence specificity between the α and the β arms of IHF could be explained by a difference in how the DNA is arranged in the complex. The fact that the interactions between IHF and DNA are asymmetric may drive the arms to making different interactions with the minor groove. The α arm of IHF might be rigidified by unique features in its amino acid sequence, such as P61 and P72, thereby making it more effective in filtering DNA sequences.

VIII. Reference

Lemberg, K., Rice, P., Swinger, K., and Zhang, Y. (2003). Flexible DNA bending in HU-DNA cocrystal structures. The EMBO Journal 22:14, 3749-3760

Nash, H.A., Mizuuchi, K., Rice, P.A., and Yang, S.-w. (1996). Crystal structure of an IHF-DNA complex: a protein-induced U-turn. Cell 87, 1295-1306

Goosen, N., and van de Putte, P. (1995). The regulation of transcription initiation by integration host factor. Mol. Microbiol. 16, 1-7

Oberto, J., and Rouviere-Yaniv, J. (1996). Serratia marcescens contains a heterodimeric HU protein like Escherichia coli and Salmonella tryphimurium. J. Bact. 178, 293-297

Zullianello, L., van Ulsen, P., van de Putte, P., and Goosen, N. (1995). Participation of the flank regions of the integration host factor protein in the specificity and stability of DNA binding. J. Biol. Chem. 270, 17902-17907

Back to Top