Lodish • Berk • Kaiser • Krieger • scott • Bretscher • Ploegh • Matsudaira

MOLECULAR CELL BIOLOGY SEVENTH EDITION

CHAPTER 3 Protein Structure and Function

Copyright © 2013 by W. H. Freeman and Company

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Figure 2.13 Overview of the cell’s principal chemical building blocks.

Macromolecules in organisms 1) Nucleic acid (핵산): 유전정보 2) Proteins (단백질): 효소, 호르몬, 막단백질, 항원, 항체 등 3) Lipids (지질): 세포막, 에너지원 4) Carbohydrates (탄수화물): 에너지원, 구조물질

Cell’s four main types of biological macromolecules The four main types of biological macromolecules—proteins, nucleic acids, polysaccharides and lipid—are all polymers composed of multiple covalently linked building block small molecules, or monomers (Figure 2-13).

Figure 3.1 Overview of protein structure and function.

Protein structure and function 1. Proteins have a hierarchical structure. A polypeptide’s linear sequence of amino acids linked by peptide bonds (primary structure) folds into helices or sheets (secondary structure) that pack into a complex three-dimensional shape (tertiary structure). Some individual polypeptides associate into multichain complexes (quaternary structure), which in some cases can be very large, consisting of tens to hundreds of subunits (supramolecular complexes). 2. Proteins perform numerous functions, including organizing in three-dimensional space the genome, organelles, the cytoplasm, protein complexes, and membranes (structure); controlling of protein activity (regulation); monitoring the Environment and transmitting information (signaling); moving small molecules and ions across membranes (transport); catalyzing chemical reactions (via enzymes); and generating force for movement (via motor proteins). These functions and others arise from specific binding interactions and conformational changes in the structure of a properly folded protein (Figure 3-1).

Figure 2.14 The 20 common amino acids used to build proteins.

Amino acid To understand the structures and functions of proteins, you must be familiar with some of the distinctive properties of the amino acids, which are determined by their side chains. The side chains of different amino acids vary in size, shape, charge, hydrophobicity, and reactivity. Amino acids can be classified into several broad categories based primarily on their solubility in water, which is influenced by the polarity of their side chains. The side chain (R group) determines the characteristic properties of each amino acid and is the basis for grouping amino acids into three main categories: hydrophobic, hydrophilic, and special (Figure 2-14). ① Amino acids with polar side chains are hydrophilic and tend to be on the surfaces of proteins; by

interacting with water, they make proteins soluble in aqueous solutions and can form noncovalent interactions with other water-soluble molecules.

② In contrast, amino acids with nonpolar side chains are hydrophobic; they avoid water and often aggregate to help form the water-insoluble cores of many proteins. The polarity of amino acid side chains thus is responsible for shaping the final three-dimensional structure of proteins.

Figure 3.2 Four levels of protein hierarchy.

Four levels of protein hierarchy We consider the architecture of proteins at four levels of organization: primary, secondary, tertiary, and quaternary (Figure 3-2).

Figure 3-5 Molecular Biology of the Cell (© Garland Science 2008)

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Figure 3-6a Molecular Biology of the Cell (© Garland Science 2008)

Protein tertiary structure is determined by its amino acid sequence. Christian Boehmer Anfinsen (1916~1995) 리보핵산가수분해효소의 1차구조 결정 및 입체구조 해석 S.무어, W.H.스타인과 함께 1972년 노벨화학상 수상

Protein tertiary structure is determined by its amino acid sequence In the mid 1950's Anfinsen began to concentrate on the problem of the relationship between structure and function in enzymes. On the basis of studies on ribonuclease with Sela and White, he proposed that the information determining the tertiary structure of a protein resides in the chemistry of its amino acid sequence. Ribonuclease A 3o structure could be destroyed by urea and mercaptoethanol, a process called denaturing. It would refold spontaneously, recovering all its activity, when these denaturing agents were removed. The denatured protein can spontaneously regenerate its native conformation. Protein folding has been studied in a test tube by using highly purified proteins. A protein can be unfolded, ordenatured, by treatment with certain solvents, which disrupt the noncovalent interactions holding the folded chain together. This treatment converts the protein into a flexible polypeptide chain that has lost its natural shape. When the denaturing solvent is removed, the protein often refolds spontaneously, or renatures, into its original conformation , indicating that all the information needed for specifying the three-dimensional shape of a protein is contained in its amino acid sequence (Figure 3-6a Molecular Biology of the Cell).

Heat shock proteins (HSPs): chaperones that facilitate protein folding and assembly

J Biol Chem. (1990) 265(21):12111-12114

Figure 3.16 (a) Molecular chaperone-mediated protein folding.

Chaperones 1. Molecular chaperones, which bind to a short segment of a protein substrate and stabilize unfolded or partly folded proteins, thereby preventing these proteins from aggregating and being degraded. Another family of heat shock proteins (with subunit moleculelar weights of about 60,000) also forms complexes with polypeptides and has ATPase activity (Table II). But in contrast to the postulated unfolding and disassembly role of most forms of hsp70, hsp60s participate in the folding and assembly of polypeptides. Based on this property, they have been referred to as chaperonins (J Biol Chem. (1990) 265(21):12111-12114). 2. The heat-shock protein Hsp70 in the cytosol and its homologs are molecular chaperones. Hsp70 and its homologs are the major chaperones in all organisms. These molecular chaperones transiently bind to a nascent polypeptide as it emerges from a ribosome or to proteins that have otherwise unfolded (Figure 3-16 (a)). ① In the Hsp70 cycle, an unfolded protein substrate binds in rapid equilibrium to the open

conformation of the substrate-binding domain (SBD) of the monomeric Hsp70, to which an ATP is bound in the nucleotide-binding domain (NBD) (step 1).

② The substrate binding pocket is shown as a green patch on the substrate-binding domain. Co-chaperone accessory proteins (DnaJ/Hsp40) stimulate the hydrolysis of ATP to ADP and conformational change in Hsp70, resulting in the closed form, in which the substrate is locked into the SBD; here proper folding is facilitated (step 2).

③ Exchange of ATP for the bound ADP, stimulated by other accessory co-chaperone proteins (GrpE/BAG1), converts the Hsp70 back to the open form (step 3), releasing the properly folded substrate (step 4).

Figure 3.16 (b) Molecular chaperone-mediated protein folding.

HSP90 3. Hsp90 proteins are dimers, whose monomers contains an N-terminal NBD domain, a central substrate (client) binding domain (SBD), and a C-terminal dimerization domain (Figure 3-16 (b)). ① The Hsp90 cycle begins when there is no nucleotide bound to the NBD and the dimer is in a very

flexible, open (Y-shaped) configuration that can bind substrates (step 1). ② Rapid ATP binding leads to a slow conformational change in which the NBDs dimerize and the

SBDs move together into a closed conformation (step 2). ③ ATP hydrolysis results in folding of the client and client protein release (step 3 and 4). ④ The ADP-bound form of Hsp90 can adopt several conformations, including a highly compact form.

Release of ADP regenerates the initial state, which can then interact with additional clients (step 4).

Figure 6-87 Molecular Biology of the Cell (© Garland Science 2008)

Figure 3.17 Chaperonin-mediated protein folding.

Chaperonin 1. Chaperonins, which form small folding chambers into which all or part of an unfolded protein can be sequestered, giving it time and an appropriate environment to fold properly. 2. The GroEL-GroES folding cycle (Figure 6-87 Molecular Biology of the Cell, Figure 3-17). ① A partly folded or misfolded polypeptide enters one of the folding chambers (step 1). ② The second chamber is blocked by a GroES lid. Each ring of seven GroEL subunits binds seven

ATPs, hydrolyzes them, and releases the ADPs in a set order coordinated with GroES binding and release and polypeptide binding, folding, and release. The major conformational changes in the GroEL rings that take place control the binding of the GroES lid that seals the chamber (step 2).

③ The polypeptide remains encased in the chamber capped by the lid, where it can undergo folding until ATP hydrolysis, the slowest, rate-limiting step in the cycle (t1/2~10s) (step 3), induces binding of ATP and a different GroES to the other ring (transient intermediate shown in brackets).

④ This then causes the GroES lid and ADP bound to the peptide-containing ring to be released, opening the chamber and permitting the folded protein to diffuse out of the chamber (step 4). If the polypeptide folded properly, it can proceed to function in the cell. If it remains partially folded or misfolded, it can rebind to an unoccupide GroEL and the cycle can be repeated.

Figure 6-90 Molecular Biology of the Cell (© Garland Science 2008)

The proteasomal degradation system degrades unneeded or damaged proteins: a highly specific process and involved in many cellular processes, including cell cycle and gene expression.

The ubiquitin-proteasome system

Annu Rev Biochem. (2012) 81:177-201

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Annu Rev Biochem. (2012) 81:177-201

Figure 3.29 Ubiquitin- and proteasome-mediated proteolysis.

The Nobel Prize in Chemistry 2004: discovery of ubiquitin-mediated protein degradation

The ubiquitin-proteasome system 1. Eukaryotic cells have several intracellular proteolytic pathways for degrading misfolded or denatured proteins, normal proteins whose concentration must be decreased, and extracellular proteins taken up by the cell. 2. Architecture of the 26S proteasome. The proteasome is a self-compartmentalized protease that consists of three main subcomplexes: a 20S core particle (CP) that houses three distinct proteolytic activities in its inner core, a 19S lid that recognizes and deubiquitylates substrates, and a 19S base structure that uses the energy of ATP hydrolysis to unfold proteins and pass them into the 20S CP for destruction. 26S proteasomes can be capped at one or both ends by a 19S complex (Annu Rev Biochem. (2012) 81:177-201, Figure 6-90 Molecular Biology of the Cell ) 3. Distinct from the lysosomal pathway are cytosolic mechanisms for degrading proteins. Chief among these mechanisms is a pathway that includes the chemical modification of a lysine side chain by the addition of ubiquitin, a 76-residue polypeptide, followed by degradation of the ubiquitin-tagged protein by a specialized proteolytic machine. Ubiquitination is a three-step process (Figure 3-29) : ① Activation of ubiquitin-activating enzyme (E1) by the addition of a ubitiquin molecule, a reaction that requires ATP ② Transfer of this ubiquitin molecule to a cysteine residue in ubiquitin-conjugating enzyme (E2) ③ Formation of a peptide bond between the ubiquitin molecule bound to E2 and a lysine residue in the target protein, a

reaction catalyzed by ubiquitin ligase (E3) ④ This process is repeated many times, with each subsequent ubiquitin molecule being added to the preceding one. The

resulting polyubiquitin chain is recognized by a proteasome, another of the cell’s molecular machines. The numerous proteasomes dispersed throughout the cell cytosol proteolytically cleave ubiquitin-tagged proteins in an ATP dependent process that yields short (7- to 8-residue) peptides and intact ubiquitin molecules.

4. Ubiquitin conjugation factor E4 is involved in N-terminal ubiquitin fusion degradation proteolytic pathway. E4 binds to the ubiquitin moieties of preformed conjugates and catalyses ubiquitin chain assembly in conjunction with E1, E2, and E3 (http://www.ebi.ac.uk/interpro/entry/IPR019474). A destruction element in the substrate (a degron) is activated, often by phosphorylation in response to a specific signal. The degron is then bound by a ubiquitin (Ub)-protein ligase (E3), which acts in conjunction with a Ub-activating enzyme (E1) and Ub-conjugating enzyme (E2) to transfer Ub (green circle) to the substrate (SUBS), typically at a lysine residue. Repeated rounds of this process, perhaps catalyzed by a Ub chain elongation factor (E4), give rise to a polyubiquitylated substrate. Ubiquitylation can be opposed by the action of deubiquitylating enzymes (DUbs), which can remove all Ub or trim the length of the Ub chain (Annu Rev Biochem. (2012) 81:177-201).

Figure 6-88 Molecular Biology of the Cell (© Garland Science 2008)

Figure 3.18 Alzheimer’s disease is characterized by the formation of insoluble plaques composed of amyloid protein.

Insoluble protein plaques Recent evidence suggests, however, that a protein may fold into an alternative three-dimensional structure as the result of mutations, inappropriate post-translational modification, or other as-yet-unidentified reasons. Such “misfolding” not only leads to a loss of the normal function of the protein but also marks it for proteolytic degradation. The subsequent accumulation of proteolytic fragments contributes to certain degenerative diseases characterized by the presence of insoluble protein plaques in various organs, including the liver and brain (Figure 6-88 Molecular Biology of the Cell). Some neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease in humans and transmissible spongiform encephalopathy (“mad cow” disease) in cows and sheep, are marked by the formation of tangled filamentous plaques in a deteriorating brain (Figure 3-18).

Regulation of protein activity : ON-OFF system 1. Post-translational modification 1) phosphorylation 2) acetylation and methylation 3) hydroxylation 4) carboxylation 5) glycosylaton 6) sumoylation & ubiquitination 2. Interaction with cofactor or ligand 3. Protein-protein interaction

Figure 6-82 Molecular Biology of the Cell (© Garland Science 2008)

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Figure 3.33 Regulation of protein activity by phosphorylation and dephosphorylation.

The Nobel Prize in Physiology or Medicine 1992: discoveries concerning reversible protein phosphorylation as a biological

regulatory mechanism

Regulation of protein activity by phosphorylation and dephosphorylation In addition to exploiting the noncovalent regulators described above, cells can use covalent modifications to regulate the intrinsic activity of a protein. One of the most common covalent mechanisms for regulating protein activity is phosphorylation, the reversible addition of phosphate groups to hydroxyl groups on the side chains of serine, threonine, or tyrosine residues. Phosphorylation is catalyzed by enzymes called protein kinases, while the removal of phosphates, known as dephosphorylation, is catalyzed by phosphatases. The counteracting activities of kinases and phosphatases provide cells with a “switch” that can turn on or turn off the function of various proteins. Phosphorylation changes a protein’s charge and can lead to a conformational change that can significantly alter ligand binding or other features of the protein, causing an increase or decrease in its activity (Figure 3-33).

Figure 2.15 Common modifications of amino acid side chains in proteins.

Regulation of protein activity by post-translational modification The difference is due to the chemical modifications of some of amino acids after they are incorporated into protein by the addition of acetyl groups (CH3CO) and a variety of other chemical groups. These modified residues and numerous others are formed by addition of various chemical groups to the amino acid side chains during or after synthesis of a polypeptide chain (Figure 2-15).

Figure 3.31 Conformational changes induced by Ca2+ binding to calmodulin.

Regulation of protein activity by interaction with cofactor or ligand 1. Two additional allosteric ligands, Ca2+ and GTP, act through two types of ubiquitous proteins to regulate many cellular processes. 2. Calmodulin-Mediated Switching The concentration of Ca2+ free in the cytosol is kept very low (≈10-7 M) by membrane transport proteins that continually pump Ca2+ out of the cell or into the endoplasmic reticulum. This rise in cytosolic Ca2+ is sensed by Ca2+-binding proteins, particularly those of the EF hand family, all of which contain the helixloop-helix motif discussed earlier. The prototype EF hand protein, calmodulin, is found in all eukaryotic cells and may exist as an individual monomeric protein or as a subunit of a multimeric protein. The binding of Ca2+ to calmodulin causes a conformational change that permits Ca2+ /calmodulin to bind various target proteins, thereby switching their activity on or off. Calmodulin and similar EF hand proteins thus function as switch proteins, acting in concert with Ca2+ to modulate the activity of other proteins (Figure 3-31).

Figure 15.6 GTPase switch proteins cycle between active and inactive forms.

Figure 3.32 The GTPase switch .

Regulation of protein activity by the GTPase switch 3. Another group of intracellular switch proteins constitutes the GTPase superfamily. As the name suggests, these protein are enzymes, GTPase, that can hydrolyze GTP (guanosine triphosphate) to GDP (guanosine diphosphate). All the GTPase switch proteins exist in two forms, or conformations: an active (“on”) form with bound GTP that can influence the activity of specific target proteins to which they bind and an inactive (“off”) form with bound GDP (Figure 15-6, 3-32).

① The switch is turned on, that is, the conformation of the protein changes from inactive to active,

when a GTP molecule replaces a bound GDP in the inactive conformation. ② The switch is turned off when the relatively slow GTPase activity of the protein hydrolyzes bound

GTP, converting it to GDP and leading the conformation to change to the inactive form.

Protein-protein interaction

Molecular Biology of the Cell (4th ed)