Conformational Changes between Open Form and Closed Form

Conformational change between the open and closed forms is seen in the D`1 and D`2

helices, but, because of disorder in the peptide chain, no density was seen in the closed form for residues 169–178, which contain helix D`3. Interestingly, residues

148–179 contain the D`1, D`2 and D`3 helices, which represent insertions into the basic

αβ hydrolase fold.

The conformational change in this region appears to be driven mainly by a rotation and twisting movement of the D`2 helix towards the active site of the enzyme. As an

example of the magnitude of this motion, the α carbon of Asn 159 moves 10.23 Å,

bringing the sidechain of the residue over the top of the P2 binding site.

Figure 2.5 shows the differences in Phi and Psi between residues 147- and 167, and the sum of these differences. From this plot, it can be seen that there are three regions that flex during the conformational change, allowing movement to occur. These are residues 151–152, 154–159 and 164–166. It is perhaps unsurprising that three of the six residues that have significant mainchain torsional differences are glycine, which

Figure 2.5 Graphical representation of Kleywegt (Ramachandran difference) plot (Kleywegt 1996) . Phi and Psi differences were calculated using (Phiclosed –

Phiopen) and (Psiclosed – Psiopen). The Kleywegt series was calculated as the

hypotenuse of Phi and Psi differences using √((Phiclosed – Phiopen)2 + (Psiclosed –

Psiopen)2). -250 -200 -150 -100 -50 0 50 100 150 200 250 300 146 148 150 152 154 156 158 160 162 164 166 168 Residue number D e g re e s Phi diff Psi diff Kleywegt

Figure 2.6 Displacement of α carbons by residue during conformational change in the mobile loop.

0 2 4 6 8 10 12 14 145 150 155 160 165 170 Residue D is p la c e m e n t (A )

Although the residues in between these flexible regions appear to have relatively stable conformations, the interactions made by several of the sidechains change during the conformational change (Figure 2.7). On the D` insertion, residues Trp 163 and Phe 167 interact laterally. The resultant aromatic plane is capped by the

sidechains Ile 166 and Tyr 162, which interact with it via van der Waals’ interactions. The sidechain of Phe 173 is found underneath the plane, and also interacts with Phe 167 and with Phe 149, which interacts with Trp 163, all through van der Waals’ interactions. The front of the plane interacts with Phe 79, Tyr 125 and Val 81, and, together, these form quite an extensive hydrophobic pocket.

Figure 2.7 Stereoviews of changes in the P2 pocket and cavity during

conformational change from the open state (green) to the closed state (yellow). Key residue sidechains are shown as sticks, with the range of movement tracked by dotted lines. Upper panel: View into substrate-binding cleft. Lower panel: View upwards from the opposite direction, to convey how great the rearrangements of residues are in this region.

178 171 177 Ser 121 171 177 182

The conformational change to the closed state causes both Phe 167 and Trp 163 to rotate away from the hydrophobic pocket, outwards towards the protein surface. During this change, the sidechain of Tyr 162, which is found stacked on top of the sidechain of Trp 163 in the open state, rotates into the same position and plane that was previously occupied by Trp 163. Phe 156 moves from a position on the outside of the D`1 helix to form a π stack with Tyr 162. These two sidechains are bracketed on

one side by the sidechain of Met 152, on the other side by Val 146 and Met 122 and on the top by Met 46. Phe 203 also rotates towards Phe 156, where the Cζs of each

sidechain are within van der Waals’ distance of each other (3.64 Å).

The open-to-closed conformational change also causes the D`1 helix to partially

unwind so that the helical region spans only residues 152–156 (compared with

residues 152–158 in the open form). The D`2 helix appears to stay intact, undergoing a

rotation of ≈ 100o as a whole. As the D`3 helix (included in the unbuilt region from

residues 169 to 178) is disordered in the closed state, it is not clear what kind of conformational change it undergoes. What can be seen is that residues Glu 179 and Gly 168, which are defined with good electron density, change their position to become separated by a distance of 7 Å. Such a change in position can occur only if

the D`3 helix unwinds to form a loop. Disorder caused by the enzyme changing from

one conformation to the other may be responsible for the absence of strong electron density in this region of the closed structure.

A displacement between residues 74 and 77 in the loop formed by residues 74–91 is also seen, between strand 2 and helix A. Density for the peptide chain is not visible for residues 78–89, suggesting disorder. In the open form, this region is closely associated with the D`2 helix, making it likely that the changes observed are linked to the conformational shift of the D` helices. In the open state, the loop comprising residues 74–91 appears to be anchored by the interaction of Phe 79 and Val 81 with Trp 163 and Phe 167, as described above. Some stabilisation of the 83–91 loop also appears to be due to Tyr 85, the sidechain of which is found in a π-stacked interaction

cooperative substrate binding. In such a scenario, substrate binding to one monomer would cause it to adopt an open conformation. The loops that are disordered during the closed state would become ordered, moving Tyr 85 into position to interact with its counterpart on the other dimer, and, as a consequence, pulling the 74–90 loop into the conformation found in the open enzyme. This stabilisation would, in turn, induce the 147–179 loop into the alternative conformation seen in the open state, forming the substrate-binding pocket. Whether cooperative binding occurs in EstA is unclear as the appropriate kinetic work has not been carried out. It should also be noted, that, as the natural substrates for EstA are not known, it is impossible at this time to verify that the enzyme is an allosteric enzyme.