Fundamentals of Molecular Biology. Part 1: Cell Molecular Biology - A. N. Ogurtsov 2011

Molecular basis of protein function
Molecular recognition

Even before the atomic Structure OF THE first Biomolecules was determined, physicist H.R. Crane formulated two principles governing macromolecular recognition in self-assembling systems.

First, to ensure high Specificity, numerous weak interactions must form between the interfacing surfaces of both interacting entities.

This principle is not immediately obvious. It might seem preferable to use a single, strong bond instead. Indeed, relying on one or a few robust bonds would provide high stability, but it would fail to ensure specificity. Because the identical spatial arrangement of just two (or several) atoms can (by chance) occur in a generally arbitrary combination of interacting particles, this increases the risk of forming accidental, erroneous complexes. In contrast, employing an entire array of weak pairwise interactions guarantees specificity, as each pair of interacting atoms contributes to the net interaction, thereby providing the necessary binding strength between two biomolecular objects.

Second, the interacting surfaces of the two biomolecules must be geometrically (topologically) similar, or more precisely, complementary to each other.

It is this exact complementarity that ensures the proper relative positioning of atoms to form a system of multiple interactions. In biological molecules, this complementarity encompasses both geometric complementarity—where protrusions On the surface of one molecule precisely match cavities on The surface of another—and "chemical complementarity," in which the precise atoms and functional groups that form Hydrogen Bonds or electrostatic attractions are positioned correctly. Such complex geometric-chemical complementarity is crucial for achieving specific interactions.

Thus, a protrusion on one surface must not only fit snugly into a cavity on the other, complementary surface, but it will also prevent binding with any surface that lacks a corresponding cavity. Adding a single methyl group to act as such a protrusion on one of the interacting surfaces may suffice to preclude macromolecule binding. For instance, the methylation of restriction sites protects bacterial DNA from the action of restriction Enzymes.

On the other hand, if for any reason one of the numerous hydrogen bonds fails to form, this does not fatally destabilize the intermolecular binding process. As a rule, Proteins possess unique interfacing surfaces that ensure binding exclusively with the intended partner while precluding interactions with any other competing molecules.

Biomolecules interact via developed interface surfaces, forming a complex network of weak interactions distributed along perfectly complementary surfaces. Molecular recognition predominantly relies on non-covalent interactions, whereas covalent bonding occurs quite rarely. Covalent bonding is reserved exclusively for The formation of robust (permanent) structures. Instead of covalent bonds, a combination of hydrogen bonds, Electrostatic Interactions between charged atoms, and hydrophobic interactions is typically employed.

Crane's two principles hold true across hundreds of studied natural systems. It is particularly important to consider these principles when examining the Structure and function of enzyme active sites and the complementary interface regions of proteins such as IMMUNOGLOBULINS.

The functioning of virtually all proteins relies on their ability to bind other molecules—ranging from simple ions to macromolecules such as proteins and Nucleic Acids, known as ligands—with a high degree of specificity.

Enzymes bind exclusively to their specific substrates (for example, hormone Membrane Receptors bind to specific Hormones).

The binding of protein factors to specific nucleotide sequences within the DNA molecule serves as the primary mechanism for regulating Gene Expression.

Ligand binding frequently induces a conformational change in the protein, and this conformational transition is an integral component of the complex mechanism of protein activity.

Specificity refers to the property of a protein to bind preferentially to a particular molecule.

Affinity is the quantitative measure that characterizes the strength of protein-ligand binding.

The more precisely the ligand and the ligand-binding site on the protein molecule are matched both geometrically and chemically, the higher the specificity and affinity of the protein-ligand interaction.

6.6.1. Antibodies. The ability of proteins to distinguish among different molecules is arguably most pronounced in antibodies (immunoglobulins)—Blood proteins produced by animals in response to the appearance of Antigens, such as infectious agents (e.g., Bacteria and Viruses) or specific foreign substances (e.g., proteins or Polysaccharides in plant pollen) (Figure 125).

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Figure 125 - Antibody: a - molecular diagram; b - antibody specificity; c - antibody structure; d - B Cells; 1 - antigen; 2 - H chain; 3 - L chain; 4 - CDR

In response to the appearance of antigens, the body produces a vast array of antibodies, each capable of binding to distinct, specific protruding regions—known as antigenic determinants, or epitopes—on the antigens.

Antibodies act as specific sensors for antigens, forming antibody-antigen complexes that trigger a cascade of defensive reactions within the cells of The Immune System.

All antibodies are Y-shaped and composed of two identical "heavy" (H) chains and two identical "light" (L) chains (Figure 125). Each "arm" of the antibody molecule is formed by one H chain and one L chain linked together by disulfide bridges. At the tip of each arm lie six polypeptide loops that form the antigen-binding site—the complementarity-determining region (CDR)—directed toward the antigen epitope.

The structure and sequence of these six loops vary extraordinarily among different antibodies, ensuring the high specificity of Antibody Binding to its complementary antigen. The interaction between the antibody CDR and the antigen epitope is complementary in every respect: the surface Topography of the epitope precisely matches the surface topography of the CDR (Figure 125(b)), and this consummate surface contact, stabilized by a network of non-covalent bonds, ensures superb specificity in antigen-antibody binding.

Antibody specificity is so exquisite that they can distinguish individual cells and, in some cases, proteins that differ by a single amino acid.

Antibodies are synthesized by white Blood Cells called B lymphocytes, or B cells (Figure 125(d)). These antibodies are located on the surface of these cells and act as receptors that recognize specific antigens.

The binding of these attached antibodies of a given B Cell to the corresponding antigen stimulates the B cell to begin proliferation and active synthesis of antibodies. These antibodies are released into the Blood Plasma and share the same specificity as the "signal" antibodies on the B cell surface. The antibodies bind to infectious agents, and then such aggregates are either engulfed by phagocytes for proteolytic degradation ("cellular Digestion") or degraded in the bloodstream by specific enzymes.

During B cell proliferation, antibody binding sites may undergo modification. Daughter cells whose antibodies possess more efficient binding sites bind to antigens more frequently and tightly, thereby gaining an advantage in further GROWTH AND REPRODUCTION. Conversely, daughter cells whose binding sites bind antigens more weakly proliferate less successfully. This is how the immune system undergoes Selection and "tailoring" to xenobiotics.

According to the clonal selection theory, the extraordinary diversity of antibodies is explained by the fact that Germ Cells do not contain whole genes for light and heavy antibody chains, but rather parts or fragments of these genes. Within these cells, these parts are assembled into "cassettes"—separately with many variants for each of the three fragments of the heavy chain variable domain, separately for the light chain, separately for the constant domains of each chain, and separately for the flexible linkers connecting the antibody domains. During the formation of somatic immune cells, these gene fragments are thoroughly shuffled and, furthermore, mutate in an as-yet-unknown manner within their hypervariable regions before joining together to form complete genes for the light and heavy antibody chains.

6.6.4. Monoclonal Antibodies. B cells from an animal infected with a given antigen synthesize a multitude of different antibodies that bind to various regions (antigenic determinants or epitopes) of that antigen. In most cases, using such a heterogeneous mixture of different antibodies is impractical; it is necessary to isolate a single characteristic antibody. To achieve this, one must isolate the unique B cell that synthesizes this antibody and then culture it. However, the number of B cell divisions is limited, making it impossible to synthesize the required quantity of antibodies this way.

In the late 1970s, this problem was solved. B cells were fused with "immortal" Cancer cells. Such hybrid cells can be cultured to synthesize the desired quantity of antibodies. These antibodies are called monoclonal antibodies because they are produced by cloning identical hybrid cells. Today, monoclonal antibodies can be synthesized for virtually any conceivable antigen.

6.6.3. Enzymes. Unlike antibodies, which bind ligands and simply pass them on to Other components of the immune system, enzymes chemically alter their ligands, which in enzymatic reactions are referred to as substrates.

An enzyme is a protein that participates in a biochemical reaction and alters its rate, but does not become part of either the starting Materials or the reaction products, remaining unchanged upon the completion of the chemical reaction.

Virtually all Chemical Reactions in The Cell are catalyzed by enzymes. Much like inorganic catalysts, enzymes—since they are not incorporated into the reactants—cannot affect the driving force of a chemical reaction, which is The change in Free energy $\Delta G$ during the reaction. They cannot drive reactions for which $\Delta G > 0$ under given conditions; they can only accelerate reactions in cases where $\Delta G < 0$.

The METABOLISM/10.html">Mechanism of enzyme Action is related to the fact that they form intermediate compounds with substrates, thereby altering the reaction pathway. This new pathway is characterized by a lower energy barrier, i.e., a lower activation energy $E'_A$ compared to $E_A$ in the initial (uncatalyzed) reaction (Figure 126).

Traditional catalysts operate under extreme conditions—at high temperatures and pressures, extreme acidity, or in organic Solvents. Enzymes, however, function In aqueous solutions under physiological conditions: a Temperature of 37°C, a pressure of 1 atmosphere, and $pH = 6.5–7.5$.

Enzymes are characterized by high catalytic activity and high specificity.

The catalytic activity of an enzyme characterizes the change in The rate of a given reaction upon the Introduction of the enzyme into the reaction system. The colossal activity of enzymes allows them to accelerate reactions by a factor of $10^6$ to $10^{12}$ compared to the rate of non-enzymatic reactions under the same conditions.

Figure 126 - Effect of an enzyme on the height of the energy barrier

To date, more than 10,000 enzymes are known. Their Classification is provided in the Enzyme Catalog (EC) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology: http://www.chem.qmul.ac.uk/iubmb/enzyme/.

Enzymes ensure not only the Synthesis and degradation of macromolecules, but also cell signaling and the Transmembrane Transport of substances. Most enzymes function within the cell, but some are secreted by the cell into the extracellular space to operate in the bloodstream, the digestive tract, and even outside the Organism.

The correctness of protein chain folding is determined by how precisely the side chains (radicals) of the necessary Amino Acids are positioned in space, ensuring both the unambiguous fixation of the substrate molecule on the enzyme and the activation of the required chemical transformation.

In the native (functional) conformation of the enzyme, these amino acid radicals form the Active Site of the enzyme—the region of the enzyme molecule where substrate binding and conversion take place (Figure 127).

The catalytic sites and substrate-binding sites can either be combined into a single region or spatially separated.

Figure 127 - Formation of the enzyme active site: a - protein chain folding; b - system of hydrogen and ionic bonds between the enzyme and the substrate (cAMP)

6.6.4. Protein Kinase A. As an example, let us examine the functioning of protein kinase A (PKA), a cAMP-dependent protein kinase.

cAMP (cyclic adenosine monophosphate) is formed from ATP in a reaction catalyzed by the enzyme adenylyl cyclase, and serves as a mediator of extracellular signals within animal cells (Figure 128). In our case, cAMP activates protein kinase A.

Protein kinase A, like other protein Kinases, transfers a phosphate group (phosphorylates) from ATP to Serine, Threonine, or Tyrosine, thereby altering The activity of target proteins, including in response to external stimuli. All protein kinases belong to the same protein family; consequently, they share a similar active site structure and phosphorylation mechanism.

Figure 128 - Cyclic adenosine monophosphate (cAMP)

The active site of protein kinase A is located on the catalytic subunit, which consists of 240 Amino Acids and is called the kinase core. Consisting of two domains—a large and a small one, separated by a deep cleft (pocket)—the kinase core binds ATP and the substrate protein, and catalyses The transfer of a phosphate group from ATP to the substrate protein. The active site is formed by amino acid residues from both domains of the kinase core.

The adenine ring of ATP fits precisely into the Base of the cleft between the two domains and is secured by a "Glycine-rich loop" (or glycine lid) formed by the sequence Gly-X-Gly-X-X-Gly-X-Val (where X is any amino acid) (Figure 129).

While ATP serves as a universal substrate for all protein kinases, each protein kinase recognises its specific substrate protein via a characteristic Amino Acid Sequence. For protein kinase A, this consensus sequence is Arg-Arg-X-Ser-Y, where X is any Amino Acid and Y is a hydrophobic amino acid.

The catalytic core of protein kinase A can adopt one of two Conformations: an "open pocket" or a "closed pocket". In the "open" conformation, substrates can enter the pocket and bind to the active site.

Figure 129 - Protein kinase A: a - schematic diagram of the kinase core; b - conformations of the kinase core

Substrate binding triggers a conformational transition in the enzyme molecule: the pocket is closed by the glycine lid, and the transfer of the phosphate group from ATP to the substrate protein is activated.

This exemplifies the "induced fit" mechanism of enzyme-substrate interaction, whereby the topology of the enzyme's active site adapts to the topology of the substrate.

Following phosphorylation, the ADP and phosphorylated substrate molecules lose their affinity for the enzyme; the enzyme undergoes a conformational transition back to the "open" state, the glycine lid lifts, the pocket opens, and the modified substrates are released. Kinetic studies have shown that the movement of the glycine lid is approximately 20 times slower than the transfer of the phosphate group from ATP to the protein.

Enzymes involved in a common metabolic pathway are frequently organised into molecular assembly lines. This is achieved either by forming protein aggregates, by being anchored to a shared scaffolding protein, or by genetic fusion into a single gene, thereby yielding the subunits of a single oligomeric enzyme (Figure 130).

Figure 130 - Enzymatic molecular assembly lines: a - diffusion-based mechanism of pathway operation; b - formation of enzyme aggregates or anchoring of enzymes to a shared scaffold; c - Gene Fusion resulting in the synthesis of a single multifunctional oligomeric protein