BamHI Complexed with B-DNA

I. Introduction
II. Enzyme Structure
III. BamHI Undergoes Marked Conformational Changes Upon DNA Binding
IV. Sites of Recognition Between BamHI and DNA
V. References

Note:  This exhibit is best viewed if the cue buttons ( ) are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.

BamHI (from Bacillus amyloli) is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 b.p.) of DNA and specifically cleaving them at a target site. This exhibit focuses on the structure-function relations of BamHI as described by Newman, et al. (1995). BamHI binds at the recognition sequence 5'-GGATCC-3' , and cleaves these sequences just after the 5'-guanine on each strand. This cleavage results in "sticky ends" which are 4 b.p. long. In its unbound form, BamHI displays a central b sheet, which resides in between a helices . BamHI is an extraordinarily unique molecule in that it undergoes a series of unconventional conformational changes upon DNA recognition. This allows the DNA to maintain it's normal B-DNA conformation without distorting to facilitate enzyme binding. BamHI is a symmetric dimer . DNA is bound in a large cleft that is formed between dimers; the enzyme binds in a "crossover" manner. Each BamHI subunit makes the majority of its backbone contacts with the phosphates of a DNA half site but base pair contacts are made between each BamHI subunit and nitrogenous bases in the major groove of the opposite DNA half site. The protein binds the bases through either direct hydrogen bonds or water-mediated H-bonds between the protein and every H-bond donor/acceptor group in the major groove. Major groove contacts are formed by atoms residing on the amino-terminus of a parallel 4 helix bundle . This bundle marks the BamHI dimer interface, and it is thought that the dipole moments of the NH₂-terminal atoms on this bundle may contribute to electrostatic stabilization .

II. Enzyme Structure

Several features of the BamHI monomer structure are worthy of note. First, a central, mixed b sheet (strands b 3, b 4, b 5, b 6, b 7, and b 1) is flanked by clusters of a helices . Within the mixed b sheet, a few structural motifs can be found. An anti-parallel b meander is composed of strands 3-5 . At one end of this b meander, three catalytic residues (Asp94, Glu111, and Glu113) are found. This group resides in close proximity to a scissile phosphate group. This scissile phosphate is either positioned or excluded from the active site of the enzyme, depending on which conformation the protein adopts (not shown). The parallel b strands 5-7 form part of a mononucleotide binding fold. This fold also includes a helices 4 and 6 , which have been identified as the crossover helices. When two symmetrical subunits dimerize, the center of the BamHI complex is formed by a 4 and 6, from both the left and right subunits. Together, the four helices form a parallel, four-helix bundle . The NH₂ terminus of this bundle projects towards the major groove of DNA, and as previously noted, the BamHI enzyme undergoes marked conformational changes upon DNA binding. The means by which BamHI rearranges is unique in many ways.

III. BamHI Undergoes Marked Conformational Changes Upon DNA Binding

Upon binding to DNA, the BamHI molecule undergoes a series of unique conformational changes. The major change involves the carboxy-terminal a helices of each subunit. In the free enzyme form, these regions are ordered, but upon DNA binding, these helices unwind, taking on a partially disordered helical secondary structure, termed an arm. The minor groove of DNA accommodates the arm from the right subunit . The left subunit's arm follows the sugar-phosphate backbone of the DNA (see below). The position of each arm with respect to DNA is stabilized by a single hydrogen bond which is formed between the amino group of residue Gly 194 and the phosphate group of nucleotide T7 on DNA (shown here for the right arm) . Although the right arm fits into the minor groove, making multiple DNA contacts, the left arm contacts the DNA backbone (PO₄) directly at residue Met 198 . The arm from the left subunit displays more basic residues that face the core of the enzyme. Arg201 forms a salt bridge with the internal residue Glu69 . Lys204 is then moved close to the carbonyl group of Asn 73 . In addition, the placement of the left arm is stabilized through the close proximity of Trp 206 with Tyr 75 and Tyr 96 .

Another conformational change that occurs upon BamHI-DNA binding is a change in the quaternary structure of the enzyme. This change is marked by a rotation of the BamHI subunits, decreasing the angle between the subunits on the DNA-binding cleft side by approximately 19 degrees. This action "clamps" the DNA. The narrowed cleft that results is shown by the proximity of the Gly194s of each subunit in the enzyme bound to DNA (21 Angstroms vs. 31 Angstroms in the free enzyme) . It is predicted that this rotation enables BamHI to make many contacts with the DNA backbone which would be otherwise impossible.

Another binding-induced BamHI conformational change involves a short string of residues that connect b3 and b4. Residues Pro79-Gly91 appear disordered in the unbound form of BamHI, but upon binding to DNA, residues 81-84 become a 3A . However, residues 89-91 maintain an extended conformation. This region resides in close proximity to the sugar phosphate backbone of the DNA between Thy3 and Gua4. The presence of glycine (residues 90 and 91) provides flexibility, allowing this region to follow the DNA backbone without severe steric hindrances . It should be noted that this region also forms part of the BamHI dimer interface, in addition to a 4 and a 6 from each subunit. This region makes contacts with a 6 from each subunit.

Yet another conformational change which BamHI undergoes upon DNA binding is the structural rearrangement of residues 152-157. These make up a loop which comes before helix a 6. The rearrangement is marked by residue Arg155 swiveling its side chain a full 90 degrees relative to its position in the free enzyme . Upon rearrangement, this side chain gains the capacity to make contacts which are specific for Gua3. The conformation is stabilized by a hydrogen bond that is formed between oxygen of Arg155 and the nitrogen of Ile 117. 

IV. Sites of Recognition Between BamHI and DNA

The BamHI enzyme is capable of making a large number of contacts with DNA.  Water mediated hydrogen bonding, as well as both main-chain and side-chain interactions aid in binding of the BamHI recognition sequence.  In the major groove, the majority of enzyme/DNA contacts take place at the amino terminus of the parallel-4-helix bundle, made up of a4 and a6 from each subunit.  Although a 6  from each subunit does not enter the DNA major groove, its preceding loops interact with the outer ends of the recognition site.  Conversely, a 4 from each subunit does enter the major groove in the center of the recognition sequence.  A total of 18 bonds are formed between the enzyme and DNA across the 6 base pair recognition sequence (12 direct and 6 water mediated bonds).  As discussed above, the L and R subunits bind in a cross over manner, whereby the R-subunit of BamHI contacts the left DNA half-site of the recognition sequence.  The binding of each BamHI subunit is precisely the same as its symmetrical partner. The recognition site for BamHI has a palindromic sequence which can be cut in half for ease in showing bonds.

The enzyme loop consisting of residues 152-157 bonds with the outer G-C base pair of the BamHI recognition sequence. Arg155 L donates two hydrogen bonds to Gua4 R. The bonding of Arg155 L and Gua4 R is not in the standard conformation so Glu161 L binds to Arg155 L to stabilize this binding conformation. Asp154 L forms a hydrogen bond with Cyt9 L . Bonding between Arginine and Guanine is common in many protein/DNA interactions. [Feel free to move molecule around to see all bonds, as the next buttons will position the molecule in its proper orientation.]

For the second G-C base pair of the recognition sequence, bonding is carried out with the amino-terminus of a 4, the loop preceding a 6 (152-157), as well as a water molecule.  This sequence involves the donation of a hydrogen bond to Gua5 R from Asn116 L.  The bond derives from the terminal amino group, and the interaction is properly positioned with another hydrogen bond which is shared by an oxygen of Asn116 L and the N of the main chain (Ser118 L). A water molecule, which is held in place by a bond from Asn 116 L and two bonds from Arg 122 R, also binds to Gua 5 R. Asp 154 L binds to Cyt 8 L to satisfy the hydrogen bonding potential . [Feel free to move molecule around to see all bonds, as the next buttons will position the molecule in its proper orientation.]

In contrast to the second G-C base pair described above, the inner A-T base pair participates in only one hydrogen bond with the protein.  This bond is formed between Thy7 L and Asn116 L.  Asn 116 L, by virtue of two H-bond donations, serves to bridge Gua5 R and Thy7 L.  The adenine of this A-T base pair, rather than participating in hydrogen bonding with the enzyme, is involved in maintaining interactions with 3 highly localized water molecules, which are held in place by bonds with Asn116 L, Ser118 L, and Asn116 R, not to mention bonds with each other . [Feel free to move molecule around to see all bonds, as the next buttons will position the molecule in its proper orientation.]

nteractions in the minor groove of DNA are also present in the BamHI/DNA complex.  This bonding is carried out by the right subunit arm of the BamHI dimer, as discussed previously. The R-arm is capable of interactions with both DNA recognition half-sites.  Hydrogen bonds are formed between Asp196 R and Gua5 R, Gly197 R and Cyt8 L, and Met198 R and Thy 7 R.  In addition, van der Waals forces help to stabilize the complex .

Finally, the enzyme/DNA complex is characterized by multiple interactions between the enzyme and the sugar-phosphate backbone of DNA.  Each of the BamHI subunits make 11 direct H-bonds to the DNA backbone, in addition to ~8 water mediated interactions.  The residues that participate in these interactions are the amino-terminus of a3 (57-61), the ordered region before b4 (89-91), the amino-terminus of a 4 (111-126), the amino-terminus of a 6 (146-165), and finally, residues 193 and 194 (immediately before the refolded COOH-terminal area arm) . The two BamHI subunits overlap in contacting DNA backbone atoms of the central four nucleotides of the BamHI recognition sequence.

The catalytic mechanism of BamHI will be discussed in a subsequent exhibit.

V. References

M. Newman, T. Strzelecka,  F.D. Dorner,  I. Schildkraut, A. K. Aggarwal, Science. 269, 656 (1995)

3, California Lutheran University.

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