Which level of protein structure is the strongest?

3.2.3 Tertiary structure

Tertiary structure—the three-dimensional structure of a protein—is the next level of complexity in protein folding. Whereas individual amino acids in the primary sequence can interact with one another to form secondary structures such as helices and sheets, and individual amino acids from distant parts of the primary sequence can intermingle via charge-charge, hydrophobic, disulfide, or other interactions, the formation of these bonds and interactions serve to change the shape of the overall protein. The folding that we end up with for a given polypeptide is the tertiary structure.

Tertiary Structure Deals with the Three-Dimensional Arrangement of All of the Amino acids

The tertiary structure of proteins deals with how the regional structures are put together in space. For example, the α-helices may be oriented parallel to each other or at right angles. So the tertiary structure refers to the folding of the different segments of helices, sheets, turns, and the remainder of the protein into its native three-dimensional structure. Commonly, membrane proteins are anchored in the membrane by hydrophobic alpha helices that may be far removed in the primary sequence but closely apposed in the tertiary structure of the protein.

Structural Basis for Receptor Homodimerization

Tertiary structure plays a role in how the hormone regulates receptor activation. The hormones in this family are long chain four α-helix bundle proteins [4,17]. A notable feature of their tertiary structure is that it contains no symmetry that might support equivalent binding environments for the receptors. How the two receptors bind to the asymmetric hormone was first revealed from the crystal structure of human growth hormone bound to the extracellular domain (ECD) of its receptor (hGH-R) [8]. The structure showed that the two ECDs binding to site 2 and site 2, respectively, use essentially the same set of residues to bind to two sites on opposite faces of the hormone [8] (Fig. 1). An identical model is seen in a prolactin hormone–receptor complex [18]. This binding is characterized by extraordinary local and global plasticity at the binding surfaces. The two binding sites have distinctly different topographies and electrostatic character, leading to different affinities for the receptor ECDs (Fig. 2).

The high-affinity site, site 1, is always occupied first by ECD1 [19]. This sequence of events is required because productive binding of ECD2 at site 2 of the hormone requires additional contacts to a patch of the C-terminal domain of ECD1. The binding of ECD2 is the programmed regulatory step for triggering biological action, and it involves a set of highly tuned interactions among binding interfaces in two spatially distinct binding sites. The energetic relationships between the ECD1–ECD2 contacts and the hormone–ECD2 site 2 interactions are known to be important. However, quantitative data are few in regard to which residues are the main contributors, whether they contribute in an additive or cooperative fashion, and how the binding energy is distributed in the interfaces.

71.2.2 Tertiary and Quaternary Structures

Tertiary structure refers to the configuration of a protein subunit in three-dimensional space, while quaternary structure refers to the relationships of the four subunits of hemoglobin to each other. The hemoglobin tetramer has been shown by X-ray crystallography to be an oblate spheroid with a diameter of 5.5 nm and a single axis of symmetry. The globin chains are folded so that the four heme groups are in surface clefts equidistant from each other. The four subunits forming the tetramers are labeled α1, α2, β1, and β2. While there is no contact between the two β-chains, each α-chain touches both β-chains. Bonds across the α1β1 interface are firmer than those at the α1β2 interface, and changes from oxy- to deoxyhemoglobin involve more extensive movement at the α1β2 interface. The quaternary structure changes markedly in going from oxy- to deoxyhemoglobin, and this accounts for many of the observed changes in physical properties. Hemoglobin mutations resulting in amino acid substitutions at these points can markedly alter specific functional properties.

Structure of Chemokines

Tertiary structures of chemokines are similar because of the conserved disulfide bonds. Structural analysis of chemokines reveals a flexible N-terminal region, an N-terminal loop, three antiparallel beta sheets, and a C-terminal alpha helix (Figure 1). Chemokines are believed to interact with their receptors through two domains.19 The N-region loop of chemokines interacts with the receptor N-domain residues (receptor Site-I), and the N-terminal flexible region of the chemokine associates with the extracellular loops and/or transmembrane residues of the receptor (receptor Site-II).

Although homodimers and aggregation of chemokines are often observed in structural studies, functional analysis of chemokines by in vitro assays (calcium flux, chemotaxis, internalization of receptor) shows that at least some chemokines can function as monomers to activate their receptors.5 Nonetheless, it has been reported that oligomerization is required for binding glycosaminoglycans (GAGs), which is in turn required for recruiting leukocytes in vivo. GAGs such as heparan sulfate are highly expressed on cell surfaces and extracellular matrix, and by binding to GAGs, chemokines can form high local concentrations. Site-directed mutagenesis in CCL2, CCL3, and CCL5 showed that mutants incapable of binding GAGs or incapable of forming oligomers could not recruit leukocytes into the peritoneum of mice.2

Tertiary Structure

The tertiary structure of a protein refers to the overall three-dimensional arrangement of its polypeptide chain in space. It is generally stabilized by outside polar hydrophilic hydrogen and ionic bond interactions, and internal hydrophobic interactions between nonpolar amino acid side chains (Fig. 4-7). Additional posttranslational covalent bonds in the tertiary structure may be formed with prosthetic groups. Tertiary folding begins while the protein is being molded into its primary polypeptide sequence. It is known to be guided through this process by chaperones, which are a family of proteins that bind hydrophobic patches and prevent them from being aggregated prematurely into nonfunctional entities.

Based upon their tertiary structure, proteins are often divided into globular or fibrous types. Fibrous proteins, like α-keratin, have elongated rope-like structures that are strong and hydrophobic. Globular proteins, like the plasma proteins and the immunoglobulins, are more spherical and hydrophilic.

(iv) Short range sequence considerations

Whereas tertiary structure is largely determined by interactions occurring between amino acids that are widely separated in the amino acid sequence, secondary structuring which as we have seen, might have a nucleating effect, is affected by short range sequence considerations. Not all sequences of amino acids can fold to form a stable α-helix or β conformation since, for example, blocks of amino acids of similar charge such as closely spaced glutamate residues carrying a negative charge will repel one another and clusters of amino acids with bulky side chains may either electrostatically or physically interfere with helix formation. Furthermore proline residues which have no substituent H atom on their peptide N and whose bulky pyrrolidine side chain has a fixed orientation (Figure 27.4) cannot fit into the α-helix and usually produce a bend in the chain. Bends and loops in the folded structure may thus be determined by the position of proline residues and also of certain other amino acids such as the hydroxy amino acids, serine and threonine, and also asparagine and leucine which, owing to the bulk and shape of their side chains, tend to prevent α-helix formation if they occur close together in the chain.

Modern DNA technology is now being used to study the folding rules. The amino acid composition of a protein may be altered at a specific point or points by site-directed mutagenesis and the effect of the alteration on the tertiary structure of the protein determined.

The Significance of Protein Structure

Their tertiary structure gives proteins a very specific shape and is an important feature in the ‘lock and key’ function of enzymes, or receptor sites on cell membranes. Specificity can vary, in some cases sites can allow some variation in structure in other cases not.

This feature of ‘correct fit’ is utilized by pharmaceutical companies when creating new drugs, which are designed to have a close-enough structure to a receptor site to be able to fit into it, to either block or stimulate a reaction (see Chapter 19 ‘Pharmacodynamics: how drugs elicit a physiological effect’, p. 137).

Factored into the combination of amino acids is the fact that they have chirality (see Chapter 6 ‘Isomers’, p. 36), which also affects the shape of a receptor site and the shape into which the protein can fold. Natural amino acids are always L isomers.

12.3 Structure

The tertiary structure of Glk consists of two domains (a large and a small) (Steitz et al., 1981). Between them, there is a deep cleft, where the substrate’s binding site is formed (Fig. 12.4).

The binding of glucose causes movement of the two domains, so that they get close to the cleft. It leads to a change in the conformation of the enzyme, a phenomenon known as induced fit. The domains acquire a closed and open conformation in the presence and absence of glucose, respectively (Fig. 12.5).

The bound glucose molecule is in a chair conformation and adopts the β-anomeric configuration. The glucose molecule participates in an extensive hydrogen bonding network within the active site pocket.

The Glks contain an N-terminal ATP-binding motif denoted by the sequence LXXDXGGTNXRXXL. The PP-Glk have two putative sequences for PP-binding: (1) TXGTGIGXA and (2) SXXX-W/Y-A (Ruiz-Villafán et al., 2014).

The main amino acids binding the glucose-forming hydrogen bonds are Gly, Asn, Asp, Glu, and His in the conserved ATP-Glk (Fig. 12.6). The large number of hydrogen bonds formed between the enzyme and glucose contribute to the stability of the closed structure. The putative catalytic amino acid that acts as a base in the reaction mechanism of Glk is preserved for: Asp100 E. coli, Asp189 S. cerevisiae, Asp451 T. litoralis, Asp440 P. furiosus, Asp443 Pyrococcus horikoshii.

4 Ligand-dependent structural differences in the ECD

The tertiary structure of the ECD in the GLP-1- and exendin-4-bound forms is almost identical. However, one diverging residue in the two ligands (Val33⁎ of GLP-1 and Lys27⁎⁎ of exendin-4 (9–39)) causes a shift in the side-chain conformations of four residues in or near the binding pocket of the ECD (Fig. 9.10). In exendin-4 (9–39), Lys27⁎⁎ interacts with Glu127, and positioning of the Lys27⁎⁎ side chain appears to be guided by a hydrophobic interaction with Leu123 (Fig. 9.10A). Val33⁎ of GLP-1 cannot interact with Glu127 causing Glu127 to change rotamer conformation and point its side chain away from GLP-1.

This results in a shift in the side-chain conformations of Leu123, Arg121, and Pro119 compared to the exendin-4 (9–39)-bound structure leading to closure of an otherwise water accessible cavity (Fig. 9.10A). The GLP-1 specific conformations also affect the conserved core of the ECD by rotating the guanidine group of Arg102 and by decreasing the distance between Asp67 and Arg102 compared to the exendin-4 (9–39)-bound structure. In this way, Asp67 and Arg102 interact directly through a hydrogen bond in the GLP-1-bound structure of ECD, but indirectly via a water molecule in the exendin-4 (9–39)-bound structure (Fig. 9.10B). The functional consequences of the ligand-specific conformational differences are not known, but the ligand-specific effect of the Glu127-Ala mutation in the full-length GLP-1R and the ligand-dependent ECD side-chain conformations point to subtle differences in the binding modes of GLP-1 and exendin-4.