The tertiary structure of proteins larger than 15,000 MW is typically subdivided into distinct regions called domains. Structurally, a domain is a compactly folded region of polypeptide. For large proteins, domains can be recognized in structures determined by x-ray crystallography or in images captured by electron microscopy. Although these discrete regions are well distinguished or physically separated from one another, they are connected by intervening segments of the polypeptide chain. Each of the subunits in hemagglutinin, for example, contains a globular domain and a fibrous domain (Figure 3-7a).
at positions 20 and 24 in this 25-residue motif. (c) The parallel two-stranded coiled-coil motif found in the transcription factor Gcn4 is characterized by two a helices wound around one another. Helix packing is stabilized by interactions between hydrophobic side chains (red and blue) present at regular intervals along the surfaces of the intertwined helices. Each a helix exhibits a characteristic heptad repeat sequence with a hydrophobic residue at positions 1 and 4. [See A. Lewit-Bentley and S. Rety, 2000, EF-hand calcium-binding proteins, Curr. Opin. Struct. Biol. 10:637-643; S. A. Wolfe, L. Nekludova, and C. O. Pabo, 2000, DNA recognition by Cys2His2 zinc finger proteins, Ann. Rev. Biophys. Biomol. Struct. 29:183-212.]
A structural domain consists of 100-150 residues in various combinations of motifs. Often a domain is characterized by some interesting structural feature: an unusual abundance of a particular amino acid (e.g., a proline-rich domain, an acidic domain), sequences common to (conserved in) many proteins (e.g., SH3, or Src homology region 3), or a particular secondary-structure motif (e.g., zinc-finger motif in the kringle domain).
Domains are sometimes defined in functional terms on the basis of observations that an activity of a protein is localized to a small region along its length. For instance, a particular region or regions of a protein may be responsible for its catalytic activity (e.g., a kinase domain) or binding ability (e.g., a DNA-binding domain, a membrane-binding domain). Functional domains are often identified experimentally by whittling down a protein to its smallest active fragment with the aid of proteases, enzymes that cleave the polypeptide backbone. Alternatively, the DNA encoding a protein can be
^ FIGURE 3-7 Tertiary and quaternary levels of structure in hemagglutinin (HA), a surface protein on influenza virus. This long multimeric molecule has three identical subunits, each composed of two polypeptide chains, HA1 and HA2. (a) Tertiary structure of each HA subunit constitutes the folding of its helices and strands into a compact structure that is 13.5 nm long and divided into two domains. The membrane-distal domain is folded into a globular conformation. The membrane-proximal domain has a fibrous, stemlike conformation owing to the alignment of two long a helices (cylinders) of HA2 with P strands in HA1. Short turns and longer loops, which usually lie at the surface of the molecule, connect the helices and strands in a given chain. (b) Quaternary structure of HA is stabilized by lateral interactions between the long helices (cylinders) in the fibrous domains of the three subunits (yellow, blue, and green), forming a triple-stranded coiled-coil stalk. Each of the distal globular domains in HA binds sialic acid (red) on the surface of target cells. Like many membrane proteins, HA contains several covalently linked carbohydrate chains (not shown).
subjected to mutagenesis so that segments of the protein's backbone are removed or changed. The activity of the truncated or altered protein product synthesized from the mutated gene is then monitored and serves as a source of insight about which part of a protein is critical to its function.
The organization of large proteins into multiple domains illustrates the principle that complex molecules are built from simpler components. Like motifs of secondary structure, domains of tertiary structure are incorporated as modules into different proteins. In Chapter 10 we consider the mechanism by which the gene segments that correspond to domains became shuffled in the course of evolution, resulting in their appearance in many proteins. The modular approach to protein architecture is particularly easy to recognize in large proteins, which tend to be mosaics of different domains and thus can perform different functions simultaneously.
The epidermal growth factor (EGF) domain is one example of a module that is present in several proteins (Figure 3-8). EGF is a small, soluble peptide hormone that binds to cells in the embryo and in skin and connective tissue in adults, causing them to divide. It is generated by proteolytic cleavage between repeated EGF domains in the EGF precursor protein, which is anchored in the cell membrane by a membrane-spanning domain. EGF modules are also present in other proteins and are liberated by proteolysis; these proteins include tissue plasminogen activator (TPA), a protease that is used to dissolve blood clots in heart attack victims;
Neu protein, which takes part in embryonic differentiation; and Notch protein, a receptor protein in the plasma membrane that functions in developmentally important signaling (Chapter 14). Besides the EGF domain, these proteins contain domains found in other proteins. For example, TPA possesses a trypsin domain, a common feature in enzymes that degrade proteins.
▲ FIGURE 3-8 Schematic diagrams of various proteins illustrating their modular nature. Epidermal growth factor (EGF) is generated by proteolytic cleavage of a precursor protein containing multiple EGF domains (green) and a membrane-spanning domain (blue). The EGF domain is also present in Neu protein and in tissue plasminogen activator (TPA). These proteins also contain other widely distributed domains indicated by shape and color. [Adapted from I. D. Campbell and P Bork, 1993, Curr. Opin. Struct. Biol. 3:385.]
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