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Weak and Strong Bonds Determine Macramolecuiar Structure

FIGURE 5-24 Schematic view of how the binding of an end-product inhibitor inhibits an enzyme by causing an allosterie transformation.

effects, from increasing the affinity of the protein for a second ligand, to switching the enzymalic activity of a protein on or off. This is known as allosterie regulation ajid is a prevalent control mechanism in biological systems. "ANostery" means "other shape," and the basic mechanism is as follows; A ligand binding at one site on a protein changes the shape of that protein. As a result of that change, an active site, or another binding site, elsewhere on the protein is altered in a way that increases or decreases its activity (Figure 5-24). Examples of proteins controlled in this way range from metabolic enzymes to transcriptional regulatory proteins.

The ligand (the allosterie effector) is very often a small molecule— a sugar or an amino acid. But allosterie regulation of a given protein can also be mediated by the binding of another protein, and a very similar effect can, in some cases, be triggered by enzymalic modification of a single amino acid residue within the regulated protein. We will see examples of allosterie regulation by all three mechanisms in this section,

The Structural Basis of Allosterie Regulation Is Known for Examples Involving Small Ligands, Protein-Protein Interactions, and Protein Modification

Here we consider the detailed structural basis for three cases of allosterie regulation, in one, the DNA-binding activity of a transcriptional regulator is controlled by the binding of a small molecule to that protein. In another, we see how a protein-protein interaction, and a protein phosphorylation event, can mediate allosterie regulation of an enzyme involved in cell division.

Small Molecule Effector: Lac Repressor Regulation by Allolactose

The lad gene of E. ro/j encodes the lactose repressor (Lac). This protein (about which we will learn more in Chapter 16) is controlled allosteri-cally—indeed, it was one of the earliest characterized examples of an allosterically controlled DNA-binding protein. The protein is involved in gene regulation, and, when bound to DNA, it prevents transcription of the genes required for the cell to use the sugar lactose as a carbon a

enzyme-substrate complex

Aliosleiy: Regulation of a Protein's Function by Changing Its Shape 89

source. However, when lactose is present in the environment, a specific form of this sugar (p-l-6-allolactose) induces expression of the lactose genes. The alJolactose inducer functions by directly binding to the Lac repressor protein and destabilizing its interaction with DNA.

Structural analysis reveals that the Lac repressor changes shape upon inducer binding. (Those structural studies used the artificial inducer molecule isopropybp-D-thiogalactoside [EPTGf.) This change in shape, in (urn, explains how the DNA-binding activity of the protein is weakened. Lac repressor is a large protein (a tetramer of 155 kDa) and contains distinct domains involved in DNA binding, protein multimeriza-tion, and inducer binding. The very N-terminal region of the protein (amino acids 1 to 45) is a helix-turn-helix motif that specifically binds the DNA major groove within the control region of the promoter, as we have seen in the case of \ repressor. Adjacent to this region is an additional helix, known as the hinge heiix, that makes minor groove contacts. The inducer-binding pocket, in contrast, is in the middle of the large core domain (composed of residues 62-333].

Comparing the DNA-bound structure of Lael with thai of the protein-free from DNA (and bound to inducer) provides a picture of why these two states are essentially mutually exclusive. Binding of inducer causes a distortion in the disposition of the N-terrninal half of the large core domain. This conformational change, in turn, disrupts the structure of the hinge helix, which weakens DNA binding; the structure of the adjacent helix-turn-helix domain is rendered more flexible as well, a change likely to lower the protein's affinity for its specific DNA site (Figure 5-25).

The allosteric modification of the enzyme aspartate transcar-bamoylase by its ligand, CTP, provides another example of a small molecule effector (Figure 5-26). In that case the ligand induces a well-characterized change tn protein tertiary structure.

Protein Effector: Cdk Activation by Cyctin We now turn to a case of allosteric regulation of an enzyme by the interaction between that enzyme and a regulatory protein. The enzyme (called Cdk2) is a member of a family of kinases known as cyelin-dependent kinases (Cdks) that regulate progression through the ceil cycle. It is inactive until complexed with a regulatory protein called a eycliil- Binding of that second protein induces a conformational change that alters the structure of Cdk2 around its active site, partially activating its function. Further conformational changes induced by phosphorylation of a specific threonine residue nearby activate the enzyme further (see below).

The structural details of the allosteric event mediated by cyclin binding have been established. The structure of Cdk2, free from cyclin, looks very like that of other kinases. Two elements of Cdk2 structure are critical for its regulation: an ol helix, called the PSTAIRE helix, anil a flexible loop, called the T loop. These are both located near the kinase active site.

Cyclin binding induces allosteric changes in the location of the T loop and PSTAIRE helix of Cdk2 (Figure 5-27). In the absence of a bound cyclin, the loop is located at the entrance to the active site and the helix is well away from that site. In this conformation, a glutamate residue critical to catalysis is held outside the active site. Binding of the cyclin results in the movement of the helix into the active: site, allowing the critical glutamate residue to take part in catalysis. Cyclin binding also moves the loop away from the entrance of the active site, allowing access of the protein substrate.

FIGURE 5-25 Allosteric changes of Lac repressor liach part of the ftgure shows a dimer of Lac repressor, (a) The left side of the ftgure shows the dimer of the inducer-Lac repressor complex. Binding of inducer causes a change in the Structure that reduces affinity of repressor for the operator (b) The right side of the figure shows the dimer in the absence o( inducer. In this case, the hinge helices form and the N-ierminal domain makes contact with the operator sequence. (Source: Adapted from Lewis et fll. 1996 Science 27): 6, fig 12. Copy right © 1996 Amentim Association for the Advancement of Science. Used with permission.)

FIGURE 5-25 Allosteric changes of Lac repressor liach part of the ftgure shows a dimer of Lac repressor, (a) The left side of the ftgure shows the dimer of the inducer-Lac repressor complex. Binding of inducer causes a change in the Structure that reduces affinity of repressor for the operator (b) The right side of the figure shows the dimer in the absence o( inducer. In this case, the hinge helices form and the N-ierminal domain makes contact with the operator sequence. (Source: Adapted from Lewis et fll. 1996 Science 27): 6, fig 12. Copy right © 1996 Amentim Association for the Advancement of Science. Used with permission.)

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