Protein structure and activity can be controlled by binding to ligands that may take a wide variety of molecular forms. This mode of regulation can provide a mechanism for sensing changes in the cell or environment: changes in the concentration of a ligand result in changes in binding, which are then transduced into a change in protein activity. For example extracellular receptors undergo changes in their conformation and/or oligomerization when they bind their cognate hormones or extracellular ligands. Ligand-induced conformational changes can result in either an increase or a decrease in protein activity, depending on the specific protein. Many proteins undergo changes in structure and activity that are dependent on pH–these changes result from binding or dissociation of one of the simplest types of ligands, protons. In signal transduction, one of the most common means of ligand-induced regulation is through the generation or release of second messengers, small molecules such as calcium (Ca²⁺ ions) and cyclic AMP (cAMP) that can modulate the activity of a broad range of downstream targets. We discuss this class of signaling molecules in Chapter 6. Here we describe the mechanisms of protein conformational change induced by Ca²⁺ ions and cAMP.

The protein calmodulin (CaM) is an example of a calcium-binding sensor–upon binding calcium it undergoes conformational changes that allow it to regulate the activity of a range of downstream effector proteins. Such a mechanism is useful for coupling a single input signal (change in Ca²⁺ ion concentration) to wholesale and rapid changes in many different cellular activities, including transport through channels, enzyme action, and the activity of other signaling molecules.

The concentration of calcium in extracellular fluids is approximately 10^-3 M, while its cytosolic concentration is actively maintained at ~10^-7 M by membrane pumps. Thus the influx of Ca²⁺ ions from the environment or release from intracellular stores such as the endoplasmic reticulum causes a very rapid and dramatic increase in cytoplasmic calcium concentration, which has been widely exploited for signal transduction. Some proteins, such as those involved in muscle contraction, directly bind to and sense Ca²⁺. However, in other cases Ca²⁺ is sensed through intermediate calcium sensors such as calmodulin.

Calmodulin is a compact protein containing four calcium-binding sites with a characteristic structural motif known as the EF hand which is found in a large number of calcium binding proteins and is indicative of regulation by calcium. The affinity for calcium for the EF hand sites in calmodulin ranges from 5 × 10^-7 to 5 × 10^-6 M, ideally suited for sensing increases in calcium above the resting intracellular level (~10^-7 M). The conformation of CaM changes dramatically upon calcium binding (Figure 5-1.1), altering its affinity for binding partners that include protein kinases and phosphatases, adenyl cyclases, transcriptional regulators, membrane channels and pumps, and others. The specific modes of their regulation by CaM binding are as diverse as the binding partners themselves. In the most familiar cases (for example, Ca²⁺/CaM-dependent protein kinases, or CaM-Ks), unliganded CaM (termed apo-CaM) cannot bind the kinase, but calcium-bound CaM (Ca²⁺–CaM) binds with high affinity. Binding of Ca²⁺–CaM to the CaM-K induces conformational changes in its catalytic domain that activate it and allow it to phosphorylate substrates. For some other effectors, however, it is apo-CaM that binds to the effector, and calcium binding leads to its release. In other cases, binding of CaM to the effector leads to its inhibition, not activation.

Protein kinase A (PKA), or cyclic AMP-dependent protein kinase, is one of the best-understood examples of an enzyme whose activity is directly regulated by a second messenger. As its name suggests, the ability of PKA to phosphorylate substrates is entirely dependent on the presence of cAMP, the level of which is in turn regulated by numerous upstream signals (we describe the regulation of cyclic AMP levels in more detail in Chapter 6). Thus, the role of PKA is to convert changes in intracellular cAMP to changes the phosphorylation of substrate proteins on serine and threonine residues. PKA has two subunits, a catalytic (C) subunit, and a regulatory (R) subunit. The C subunit contains the kinase activity, but this activity is tightly controlled by its association with an R subunit. It is the R subunit that acts as a conformational switch controlled by cAMP binding (Figure 5-1.2).

In its inactive state (when levels of cAMP are low, typically below 10^-8 M), the catalytic (C) subunit exists in a 1:1 complex with the regulatory (R) subunit (there are actually several distinct R subunits with slightly different biological properties). In the absence of cAMP, the K_d for association of the two subunits is less than 10^-9 M, so essentially all of the C subunit is complexed. Binding of the R subunit sterically blocks substrates from binding to the active site of the enzyme. Thus in the basal state, when cAMP levels are low, the vast majority of PKA is in a latent state, sequestered through stable interaction with the R subunit.

The R subunit contains two distinct binding sites for cAMP which exhibit positive cooperativity. Thus at cAMP concentrations near the K_d for its binding the R subunit (between 10^-8 and 10^-7 nM), relatively small differences in cAMP levels lead to large differences in binding (see section 3-3). Once two molecules of cAMP are bound to the R subunit, a conformational change alters its binding interface with the C subunit, leading to dissociation of the C subunit in its active form to phosphorylate substrates.

Although the regulation of PKA appears straightforward, in reality the on–off switch is part of a regulatory apparatus that is considerably more complex and subtle. For example, the R subunit constitutively dimerizes, so the actual inactive complex is an R₂C₂ heterotetramer. Further, each of the R subunits interacts with specific scaffold proteins that tether the inactive complexes to specific subcellular localizations that are associated with distinct potential substrates. Once the C subunit is released, it can more freely diffuse to other parts of the cell, and for example can translocate into the nucleus and phosphorylate substrates there. Thus even this relatively simple example involves a variety of distinct protein–protein interactions, and also involves changes in protein conformation and subcellular localization.

Definitions

calmodulin (CaM): a small calcium-binding protein that confers calcium regulation on cellular signaling and effector molecules, including protein kinases and phosphatases.

CaM: see calmodulin.

PKA: see protein kinase A.

protein kinase A (PKA): a protein serine/threonine kinase that is activated by cAMP.