Peptide Bonds Restrict Possible Secondary Conformations
Free rotation is possible about only two of the three covalent bonds of the polypeptide backbone: the bond linking the α-carbon (Cα) to the carbonyl carbon (Co) and the bond linking Cα to nitrogen (see Figure 3–9). The partial double-bond character of the peptide bond that links Co to the α-nitrogen requires that the carbonyl carbon, carbonyl oxygen, and α-nitrogen remain coplanar, thus preventing rotation. The angle about the Cα—N bond is termed the phi (Φ) angle, and that about the Co—Cα bond the psi (Ψ) angle. In peptides, for amino acids other than glycine, most combinations of phi and psi angles are disallowed because of steric hindrance (Figure 5–1). The conformations of proline are even more restricted as its cyclic structure prevents free rotation of the N—Cα bond.
Ramachandran plot of the main chain phi (Φ) and psi (Ψ) angles for approximately 1000 nonglycine residues in eight proteins whose structures were solved at high resolution. The dots represent allowable combinations, and the spaces prohibited combinations, of phi and psi angles. (Reproduced, with permission, from Richardson JS: The anatomy and taxonomy of protein structures. Adv Protein Chem 1981;34:167. Copyright © 1981. Reprinted with permission from Elsevier.)
Regions of ordered secondary structure arise when a series of aminoacyl residues adopt similar phi and psi angles. Extended segments of polypeptide (eg, loops) can possess a variety of such angles. The angles that define the two most common types of secondary structure, the α helix and the β sheet, fall within the lower and upper left-hand quadrants of a Ramachandran plot, respectively (Figure 5–1).
The polypeptide backbone of an a helix is twisted by an equal amount about each α-carbon with a phi angle of approximately −57° and a psi angle of approximately −47°. A complete turn of the helix contains an average of 3.6 aminoacyl residues, and the distance it rises per turn (its pitch) is 0.54 nm (Figure 5–2). The R groups of each aminoacyl residue in an a helix face outward (Figure 5–3). Proteins contain only l-amino acids, for which a right-handed α helix is by far the more stable, and only right-handed α helices are present in proteins. Schematic diagrams of proteins represent α helices as coils or cylinders.
Orientation of the main chain atoms of a peptide about the axis of an α helix.
View down the axis of an α helix. The side chains (R) are on the outside of the helix. The van der Waals radii of the atoms are larger than shown here; hence, there is almost no free space inside the helix. (Slightly modified and reproduced, with permission, from Berg JM, Tymoczko JL, Stryer L: Biochemistry, 7th ed. Freeman, 2012. Copyright © 2012 W.H. Freeman and Company.)
The stability of an α helix arises primarily from hydrogen bonds formed between the oxygen of the peptide bond carbonyl and the hydrogen atom of the peptide bond nitrogen of the fourth residue down the polypeptide chain (Figure 5–4). The ability to form the maximum number of hydrogen bonds, supplemented by van der Waals interactions in the core of this tightly packed structure, provides the thermodynamic driving force for the formation of an α helix. Since the peptide bond nitrogen of proline lacks a hydrogen atom, it is incapable of forming a hydrogen bond with a carbonyl oxygen. Consequently, proline can only be stably accommodated within the first turn of an α helix. When present elsewhere, proline disrupts the conformation of the helix, producing a bend. Because it possesses such a small R group, glycine also frequently induces bends within α helices.
Hydrogen bonds (dotted lines) formed between H and O atoms stabilize a polypeptide in an α-helical conformation. (Reprinted, with permission, from Haggis GH, et al: Introduction to Molecular Biology Science 1964;146:1455–1456. Reprinted with permission from AAAS.)
Many α helices have predominantly hydrophobic R-groups projecting from one side of the axis of the helix and predominantly hydrophilic R-groups projecting from the other side. These amphipathic helices are well adapted to the formation of interfaces between polar and nonpolar regions such as the hydrophobic interior of a protein and its aqueous environment. Clusters of amphipathic helices can create channels, or pores, through hydrophobic cell membranes that permit specific polar molecules to pass.
The second (hence “beta”) recognizable regular secondary structure in proteins is the β sheet. The amino acid residues of a β sheet, when viewed edge-on, form a zigzag or pleated pattern in which the R groups of adjacent residues project in opposite directions. Unlike the compact backbone of the α helix, the peptide backbone of the β sheet is highly extended. But like the α helix, β sheets derive much of their stability from hydrogen bonds between the carbonyl oxygens and amide hydrogens of peptide bonds. However, in contrast to the α helix, these bonds are formed with adjacent segments of the β sheet (Figure 5–5).
Interacting β sheets can be arranged either to form a parallel β sheet, in which the adjacent segments of the polypeptide chain proceed in the same direction amino to carboxyl, or an antiparallel sheet, in which they proceed in opposite directions (Figure 5–5). Either configuration permits the maximum number of hydrogen bonds between segments, or strands, of the sheet. Most β sheets are not perfectly flat but tend to have a right-handed twist. Clusters of twisted strands of β sheet, sometimes referred to as β barrels, form the core of many globular proteins (Figure 5–6). Schematic diagrams represent β sheets as arrows that point in the amino to the carboxyl terminal direction.
Spacing and bond angles of the hydrogen bonds of antiparallel and parallel pleated β sheets. Arrows indicate the direction of each strand. Hydrogen bonds are indicated by dotted lines with the participating β-nitrogen atoms (hydrogen donors) and oxygen atoms (hydrogen acceptors) shown in blue and red, respectively. Backbone carbon atoms are shown in black. For clarity in presentation, R groups and hydrogen atoms are omitted. Top: Antiparallel β sheet. Pairs of hydrogen bonds alternate between being close together and wide apart and are oriented approximately perpendicular to the polypeptide backbone. Bottom: Parallel β sheet. The hydrogen bonds are evenly spaced but slant in alternate directions.
Examples of the tertiary structure of proteins. Top: The enzyme triose phosphate isomerase complexed with the substrate analog 2-phosphoglycerate (red). Note the elegant and symmetrical arrangement of alternating β sheets (light blue) and a helices (green), with the β sheets forming a β-barrel core surrounded by the helices. (Adapted from Protein Data Bank ID no. 1o5x.) Bottom: Lysozyme complexed with the substrate analog penta-N-acetyl chitopentaose (red). The color of the polypeptide chain is graded along the visible spectrum from purple (N-terminal) to tan (C-terminal). The concave shape of the domain forms a binding pocket for the pentasaccharide, the lack of β sheet, and the high proportion of loops and bends. (Adapted from Protein Data Bank ID no. 1sfb.)
Roughly half of the residues in a “typical” globular protein reside in a helices or β sheets, and half in loops, turns, bends, and other extended conformational features. Turns and bends refer to short segments of amino acids that join two units of the secondary structure, such as two adjacent strands of an antiparallel β sheet. A β turn involves four aminoacyl residues, in which the first residue is hydrogen-bonded to the fourth, resulting in a tight 180° turn (Figure 5–7). Proline and glycine often are present in β turns.
A β turn that links two segments of antiparallel β sheet. The dotted line indicates the hydrogen bond between the first and fourth amino acids of the four-residue segment Ala-Gly-Asp-Ser.
Loops are regions that contain residues beyond the minimum number necessary to connect adjacent regions of secondary structure. Irregular in conformation, loops nevertheless serve key biologic roles. For many enzymes, the loops that bridge domains responsible for binding substrates often contain aminoacyl residues that participate in catalysis. Helix-loop-helix motifs provide the oligonucleotide-binding portion of many DNA-binding proteins such as repressors and transcription factors. Structural motifs such as the helix-loop-helix motif or the E-F hands of calmodulin (see Chapter 51) that are intermediate in scale between secondary and tertiary structures are often termed supersecondary structures. Since many loops and bends reside on the surface of proteins, and are thus exposed to solvent, they constitute readily accessible sites, or epitopes, for recognition and binding of antibodies.
While loops lack apparent structural regularity, many adopt a specific conformation stabilized through hydrogen bonding, salt bridges, and hydrophobic interactions with other portions of the protein. However, not all portions of proteins are necessarily ordered. Proteins may contain “disordered” regions, often at the extreme amino or carboxyl terminal, characterized by high conformational flexibility. In many instances, these disordered regions assume an ordered conformation upon binding of a ligand. This structural flexibility enables such regions to act as ligand-controlled switches that affect protein structure and function.
Tertiary & Quaternary Structure
The term “tertiary structure” refers to the entire three-dimensional conformation of a polypeptide. It indicates, in three-dimensional space, how secondary structural features—helices, sheets, bends, turns, and loops—assemble to form domains and how these domains relate spatially to one another. A domain is a section of the protein structure sufficient to perform a particular chemical or physical task such as binding of a substrate or other ligand. Most domains are modular in nature, and contiguous in both primary sequence and three-dimensional space (Figure 5–8). Simple proteins, particularly those that interact with a single substrate, such as lysozyme or triose phosphate isomerase (Figure 5–6) and the oxygen storage protein myoglobin (see Chapter 6), often consist of a single domain. By contrast, lactate dehydrogenase is comprised of two domains, an N-terminal NAD+-binding domain and a C-terminal binding domain for the second substrate, pyruvate (Figure 5–8). Lactate dehydrogenase is one of the family of oxidoreductases that share a common N-terminal NAD(P)+-binding domain known as the Rossmann fold. By fusing a segment of DNA coding for a Rossmann fold domain to that coding for a variety of C-terminal domains, a large family of oxidoreductases have evolved that utilize NAD(P)+/NAD(P)H for the oxidation and reduction of a wide range of metabolites. Examples include alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, quinone oxidoreductase, 6-phosphogluconate dehydrogenase, d-glycerate dehydrogenase, formate dehydrogenase, and 3α, 20β-hydroxysteroid dehydrogenase.
Polypeptides containing two domains. Top: Shown is the three-dimensional structure of a monomer unit of the tetrameric enzyme lactate dehydrogenase with the substrates NADH (red) and pyruvate (blue) bound. Not all bonds in NADH are shown. The color of the polypeptide chain is graded along the visible spectrum from blue (N-terminal) to orange (C-terminal). Note how the N-terminal portion of the polypeptide forms a contiguous domain, encompassing the left portion of the enzyme, responsible for binding NADH. Similarly, the C-terminal portion forms a contiguous domain responsible for binding pyruvate. (Adapted from Protein Data Bank ID no. 3ldh.) Bottom: Shown is the three-dimensional structure of the catalytic subunit of the cAMP-dependent protein kinase (Chapter 42) with the substrate analogs ADP (red) and peptide (purple) bound. The color of the polypeptide chain is graded along the visible spectrum from blue (N-terminal) to orange (C-terminal). Protein kinases transfer the γ-phosphate group of ATP to protein and peptide substrates (Chapter 9). Note how the N-terminal portion of the polypeptide forms a contiguous domain rich in β sheet that binds ADP. Similarly, the C-terminal portion forms a contiguous, α helix-rich domain responsible for binding the peptide substrate. (Adapted from Protein Data Bank ID no. 1jbp.)
Not all domains bind substrates. Hydrophobic membrane domains anchor proteins to membranes or enable them to span membranes. Localization sequences target proteins to specific subcellular or extracellular locations such as the nucleus, mitochondria, secretory vesicles, etc. Regulatory domains trigger changes in protein function in response to the binding of allosteric effectors or covalent modifications (see Chapter 9). Combining the genetic material coding for individual domain modules provides a facile route for generating proteins of great structural complexity and functional sophistication (Figure 5–9).
Some multidomain proteins. The rectangles represent the polypeptide sequences of a forkhead transcription factor; 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, a bifunctional enzyme whose activities are controlled in a reciprocal fashion by allosteric effectors and covalent modification (see Chapter 19); phenylalanine hydroxylase (see Chapters 27 and 29), whose activity is stimulated by phosphorylation of its regulatory domain; and the epidermal growth factor (EGF) receptor (see Chapter 41), a transmembrane protein whose intracellular protein kinase domain is regulated via the binding of the peptide hormone EGF to its extracellular domain. Regulatory domains are colored green, catalytic domains dark blue and light blue, protein-protein interaction domains light orange, DNA binding domains dark orange, nuclear localization sequences medium blue, and transmembrane domains yellow. The kinase and bisphosphatase activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are catalyzed by the N- and C-terminal proximate catalytic domains, respectively.
Proteins containing multiple domains can also be assembled through the association of multiple polypeptides, or protomers. Quaternary structure defines the polypeptide composition of a protein and, for an oligomeric protein, the spatial relationships between its protomers or subunits. Monomeric proteins consist of a single polypeptide chain. Dimeric proteins contain two polypeptide chains. Homodimers contain two copies of the same polypeptide chain, while in a heterodimer the polypeptides differ. Greek letters (α, β, γ, etc) are used to distinguish different subunits of a hetero-oligomeric protein, and subscripts indicate the number of each subunit type. For example, α4 designates a homotetrameric protein, and α2β2γ, a protein with five subunits of three different types.
Since even small proteins contain many thousands of atoms, depictions of protein structure that indicate the position of every atom are generally too complex to be readily interpreted. Simplified schematic diagrams thus are used to depict the key features of a protein’s tertiary and quaternary structure. Ribbon diagrams (Figures 5–6 and 5–8) trace the conformation of the polypeptide backbone, with cylinders and arrows indicating regions of α helix and β sheet, respectively. In an even simpler representation, line segments that link the α carbons of each amino acid residue indicate the path of the polypeptide backbone. In order to emphasize specific structure-function relationships, these schematic diagrams often depict the side chains of selected amino acids.