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BETA-structures_Branden-Tooze

BETA-structures_Branden-Tooze - Antiparallel beta[3...

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Unformatted text preview: Antiparallel beta ([3) structures comprise the second iarge group of protein domain structures. Functionally, this group is the most diverse; it includes enzymes, transport proteins, antibodies, cell surface proteins, and virus coat proteins. The cores of these domains are built up by B strands that can vary in number from four or five to over ten. The [3 strands are arranged in a predominantly antiparallel fashion and usually in such a way that they form two [3 sheets that are joined together and packed against each other. The [5 sheets have the usual twist, and when two such twisted 13 sheets are packed together, they form a barrel—Eike structure (Figure 5.1). Antiparal— lel [3 structures, therefore, in general have a core of hydrophobic side chains inside the barrel provided by residues in the {i strands. The surface is formed by residues from. the loop regions and from the strands. The aim of this chapter is to examine a number of antiparallel B structures and demonstrate how these rather complex structures can be separated into smaller comprehensible motifs. Figure 5.1 The enzyme superoxide dismutase (SOD). SOD is a 13 structure comprising eight antiparallel [3 strands (a). In addition, SOD has two metal atoms, Cu and Zn (yellow circles), that participate in the catalytic action: conversion of a superoxide radical to hydrogen peroxide and oxygen. The eight [3 strands are arranged around the surface of a barrel, which is viewed atong the barrel axis in (b) and perpendicular to this axis in (c). [(a) Adapted from J.S. Richardson. The structure of SOD was determined in the laboratory of J.S. and DR. Richardson, Dulce University] In Chapter 2 we described the 24 different ways that two B-loop-B units can form a four-stranded B sheet. The number of possible ways to form antiparallel B—sheet structures rapidly increases as the number of strands increases. It is thus surprising, but reassuring, that the number of topologies actually observed is small and that most B structures fall into a few groups of common or similar topology. The three most frequently occurring groups— up-and-down barrels, Greek keys, and jelly roll barrels—~can all be related to simple ways of connecting antiparaliel B strands arranged in a barrel structure. Up-and-down barrels have a simple topology The simplest topology is obtained if each successive B strand is added adjacent to the previous strand until the last strand is joined by hydrogen bonds to the first strand and the barrel is closed (Figure 5.2). These are called up-and-down B sheets or barrels. The arrangement of B strands is similar to that in the DUB—barrel structures we have just described in Chapter 4, except that here the strands are antiparallel and all the connections are hairpins. The structural and functional versatility of even this simple arrangement will be illustrated by two examples. The retinal-binding protein binds retinal inside an up—and—down [3 barrel The first example is the plasma-borne retinal—binding protein, RBP, which is a single polypeptide chain of 182 amino acid residues. This protein is responsible for transporting the lipid alcohol vitamin A (retinol) from its storage site in the liver to the various vitamin—A-dependent tissues. It is a disposable package in the sense that each REP molecule transports only a single retinol molecule and is then degraded. REP is synthesized in the hepatocytes, where it picks up one molecule of retinol in the endoplasmic reticulum. Both its synthesis and its secretion from the hepatocytes to the plasma are regulated by retinol. in plasma, the 68 Figure 5.2 Schematic and topological diagrams of an up-and-down [3 barrel. The eight B strands are all antiparallel to each other and are connected by hairpin loops. Beta strands that are adjacent in the amino acid sequence are also adjacent in the three5 dimensional structure of up-and-down barrels. Figure 5.3 Schematic diagram of the structure of human plasma retinal-binding protein (REP), which is an up-and-down B barrel. The eight antiparallel B strands twist and curl such that the structure can also be regarded as two B sheets (green and blue) packed against each other. Some of the twisted B strands (red) participate in both B sheets. A retinoi molecule, vitamin A (yellow), is bound inside the barrel, between the two B sheets, such that its only hydrophiiic part (an 01-1 tail) is at the surface of the molecule. The topological diagram of this structure is the same as that in Figure 5.2. (Courtesy of Alwyn Jones, Uppsala, Sweden.) A“, RETINOL RBP-retinoi complex binds to a larger protein molecule, prealbumin, which further stabilizes it and prevents its loss via the kidney. Recognition of this complex by a cell—surface receptor causes RBP to release the retinol and, as a result, to undergo a conformational change that drastically reduces its affin— ity for prealbumin. The free RBP molecule is then excreted through the kidney glomerus, reabsorbed in the proximal tubule cells, and degraded. The structure of RBP with bound retinol has been determined in the laboratory of Alwyn Jones in Uppsala, Sweden to 2.0 A resolution. Its most striking feature is a B-barrel core consisting of eight up-and-down antiparallel B strands as shown in Figure 5.3. In addition, there is a four-turn or helix at the carboxy end of the polypeptide chain that is packed against the outside of the [3 barrel. The [3 strands are curved and twisted, and the barrel is wrapped around the retinol molecule. One end of the barrel is open to the solvent whereas the other end is closed by tight side—chain packing: the tail of the retinol molecule is at the open end of the barrel. The hydrophobic retinol molecule is packed against hydrophobic side chains from the [3 strands in the barrel’s core (Figure 5.4). The structure of the apo-forrn Where the retinol molecule is removed is also known. Surprisingly, there is almost no change in the barrel structure, and a large hole is left inside the barrel. Amino acid sequence reflects B smtcrure On a large part of the surface of RE? (the front face in Figure 5.3), side chains from residues in the B strands are exposed to the solvent. This is achieved by alternating hydrophobic with polar or charged hydrophilic residues in the Figure 5.4 The binding site for retinol inside the REF barrel is lined with hydrophobic residues. They provide a hydrophobic surrounding for the hydrophobic part of the retinol molecule. 69 strand residue air-tine acid sequence no. no. 2 4 M43 3 53—60 4 7l—78 amino acid sequences of the B strands; in other words, side chains of the B strands form the hydrophobic core of the barrel as well as part of the hydrophilic outer surface. Strands 2 3 4 of RBP clearly illustrate this arrange- ment (Figure 5.5, where the core residues are colored green). This structure also very clearly illustrates that an antiparailel 13 barrel is built up from two B sheets that are packed against each other (see Figure 5.3). Beta strands 1 2 3 4 5 6 (blue and red color) form one sheet, and strands 1 8 7 6 5 (green and red color) form the second sheet. Strands 1 5 6 thus contribute to both sheets by having sharp corners where they can turn over from one sheet to the other. The retinal-binding protein belongs to a superfamily of protein structures REP is one member of a superfamily of proteins with different functions, marginally homologous amino acid sequences, but similar three-dimensional structures. This superfamily also includes an insect protein that binds a blue pigment, biliverdin, and B-lactoglobuiin, a protein that is abundant in milk. All have polypeptide chains of approximately the same lengths that are wrapped into very similar up-and-down eight—stranded antiparallel B barrels. They all tightly bind hydrophobic ligands inside this barrel. There is a second family of small lipid-binding proteins, the P2 family, which include among others cellular retinol- and fatty acid—binding proteins as well as a protein, P2, from myelin in the peripheral nervous system. How- ever, members of this second family have ten antiparallel B strands in their barrels compared with the eight strands found in the barrels of the REF super- family. Members of the P2. family show no amino acid sequence homology to members of the RBP superfamily. Nevertheless, their three-dimensional structures have similar architecture and topology, being up-and-down B barrels. Netrmminidase folds into up-and-down [3 sheets A second example of up-and—down B sheets is the protein neuraminidase from influenza virus. Here the packing of the sheets is different from that in RBP. They do not form a simple barrel but instead six small sheets, each with four B strands, which are arranged like the blades of a six-bladed propeller. Loop regions between the B strands form the active site in the middle of one side of the propeller. Other similar structures are known with different numbers of the same motif arranged like propellers with different numbers of blades such as the G-proteins discussed in Chapter 13. Influenza virus is an RNA virus with an outer lipid envelope. There are two virai proteins anchored in this membrane, neuraminidase and hemag- glutinin. They are both transmembrane proteins with a few residues inside the membrane and a transmembrane region followed by a stalk and a head- piece outside the membrane. The heads are exposed on the surface of the viri~ on and thus provide the antigenic determinants of this epidemic virus. The function of hemagglutinin, which is glycosylated, is to mediate the binding of virus particles to host cells by recognizing and binding to sialic acid residues on glycoproteins of the cell membrane, as we shall discuss in more detail at the end of this chapter. The role of the viral neuraminidase, conversely, seems to be to facilitate the release of progeny virions from infected cells by cleaving sialic acid 70 Figure 5.5 Amino acid sequence of B strands 2 3 4 in human plasma retinal-binding pro- tein. The sequences are listed in such a way that residues which point into the barrel are aligned. These hydrophobic residues are arrowed and colored green. The remaining residues are exposed to the solvent. residues from the carbohydrate side chains both of the viral hemaggiutinin and of the glycosylated cellular membrane proteins. This helps prevent prog- eny virions from binding to and reinfecting the cells from whose surface they have just budded. From the point of view of the viruses, reinfecttng an already infected cell is, of course, a waste of time. The neuraminidase molecule is a homotetramer made up of four identi- cal polypeptide chains, each of around 470 amino acids; the exact number varies depending on the strain of the virus. If influenza virus is treated with the proteolytic enzyme pronase, the head of the neuraminidase, which is soluble, is cleaved off from the stalk projecting from the viral envelope. The soluble head, comprising four subunits of about 400 amino acids each, can be crystallized. Folding motifs form a propeller—like smtchtre in neuraminidase The structure of these tetrameric neuraminidase heads was determined in the laboratory of Peter Colman in Parkville, Australia to 2.9 A resolution. Each of the four subunits of the tetrarner is folded into a single domain built up from six closely packed, similarly folded motifs. The motif is a simple up-and— down antiparallel l3 sheet of four strands (Figure 5.6). The strands have a rather large twist such that the directions of the first and the fourth strands differ by 90°. To a first very rough approximation the six motifs are arranged within each subunit with an approximate sixfold symmetry around an axis through the center of the subunit (Figure 5.7a). These six :3 sheets are arranged like six blades of a propeller. {a} Figure 5.6 Schematic and topological diagrams of the folding motif in neuraminidase from influenza virus. The motif is built up from four antiparallel ii strands joined by hairpin loops, an up-and-down open3 sheet. Figure 5.7 The subunit structure of the neuraminidase headpiece (residues 34—469) from influenza virus is built up from six similar, consecutive motifs of four up-and— down antiparallel B strands (Figure 5.6). Each such motif has been called a propeller blade and the whole subunit structure a six-blade propeller. The motifs are connected by loop regions from [3 strand 4 in one motif to [3 strand 1 in the next motif. The schematic diagram (21) is viewed down an approximate sixfold axis that relates the centers of the motifs. Four such six-blade propeller subunits are present in each complete neuraminidase molecule (see Figure 5.8). In the topological diagram (b) the yellow loop that connects the N—terminal B strand to the first Bstrand of motif 1 is not to scale. In the folded structure it is about the same length as the other loops that connect the motifs. (Adapted from J. Varghese et 211., Nature 303: 35—40, 1983.) 71 In summary, the whole moiecule has almost 1600 amino acid residues. It is composed of four identical polypeptide chains, each of which is folded into a superbarrel with 24 B strands (Figure 5.8). These 24 B strands are arranged in six similar motifs, each of which contains four [3 strands that form the blades of a propeller—like structure. The active site is in the middle of one side of the propeller Not only are the topologies within the six [3 sheets in each subunit identical, but so are their connections to each other, with the exception of the last B sheet (see Figure 5.7b). The fourth strand of each h sheet is connected across the top of the subunit (seen in Figure 5.7a coming out of the page) to the first strand of the next sheet. The loop that connects strands 2 and 3 within the sheet is also at the top of the subunit. Furthermore, because of the approximate sixfold symmetry of the fi- sheet motifs, these 12-1oop regions, derived from the six [3 sheets, are on the same side of. the molecule, as can be seen in Figure 5.9a, where we see a single polypeptide chain (one of the four subunits) from the side of the propeller. The [3 sheets are arranged cyclically around an axis through the center of the molecule, The loop regions at the top of this barrel are exten- sive (Figure 5.9a) and together they form a wide funnei-shaped pocket containing the active site (Figure 5.91)). This is analogous to the active site formed by the loop regions at the top of the (xiii-barrel structures. Greek key motifs occur frequently in mitipamllei fl structures We saw in Chapter 2. that the Greek key motif provides a simple way to connect antiparallel Li strands that are on opposite sides of a barrel structure. We will now look at how this motif is incorporated into some of the simple antiparaliel B-barrel structures and show that an antiparaliel [3 sheet of eight strands can be built up only by hairpin and/or Greek key motifs, if the connections do not cross between the two ends of the [3 sheet. 72 Figure 5.8 Schematic view down the fourfold axis of the tetrameric molecule of neurarninidase as it appeared on the cover of Nature, May 5, 1983. r-er-Iwrgm." n Assume that we have eight antiparallel [i strands arranged in a barrel structure. We decide that we want to connect strand number n to an antipar— allel strand at the same end of the barrel. We do not want to connect it to strand number n + 1 as in the up-and-down barrels just described, nor do we want to connect it to strand number n w 1 which is equivalent to turning the up-and-down barrel in Figure 5.2 upside down. What alternatives remain? It is easy to see from Figure 5.10 that there are only two alternatives. We can connect it either to strand number n + 3 or to n — 3. Both cases require only short loop regions that traverse the end of the barrel. How do we now continue the connections? The simplest way to connect the strands that were skipped over is to join them by up—and-down connections, as illustrated in Figure 5.10- n+2 (a) - n+3 [5) 12—3 [9) Active site Figure 5.9 The six four-stranded motifs in a single subunit of neuraminidase form the six blades of a propeller-like structure. A schematic diagram of the subunit structure shows the propeller viewed from its side (a). An idealized propeller structure vieWed from the side to highlight the position of the active site is shown in (b). The loop regions that connect the motifs {red in b) in combination with the loops that connect strands 2 and 3 within the motifs (green in b) form a wide funnel-shaped active site pocket. [(51) Adapted from P. Colman et ai., Nahrre 326: 358—363, 1987.] Figure 5.10 Idealized diagrams of the Greek key motif. This motif is formed when one of the connections of four antiparallel {5 strands is not a hairpin connection. The motif occurs when strand number n is connected to strand n + 3 (a) or u - 3 (b) instead of n + 1 or n ~ 1 in an eight»stranded antiparallel [3 sheet or barrel. The two different possible connections give two different hands of the Greek key motif. In all protein structures known so far only the hand shown in (a) has been observed. 73 We have now connected four adjacent strands of the barrel in a simple and logical fashion requiring only short loop regions. The result is the Greek key motif described in Chapter 2, which is found in the large majority of antiparaliei [3 structures. The two cases represent the two possible different hands, but in all structures known to us the hand that corresponds to the case where {3 strand :1 is linked to [3 strand :1 + 3 as in Figure 5.10a is present. The remaining four strands of the barrel can be joined either by Lip-and- down connections before and after the motif or by another Greek key motif. We will examine examples of both cases. The Hrystallin molecule has two domains The transparency and refractive power of the lenses of our eyes depend on a smooth gradient of refractive index for visible light. This is achieved partly by a regular packing arrangement of the cells in the lens and partly by a smoothly changing concentration gradient of lens-specific proteins, the crystallins. There are at least three different classes of crystallins. The or and [3 are heterogeneous assemblies of different subunits specified by different genes, whereas the gamma (7) crystallins are monomeric proteins with a polypeptide chain of around 170 amino acid residues. The structure of one such y crystallin was determined in the laboratory of Tom Biundell in London to 1.9 A resolution. A picture of this molecule generated from a graphics display is shown in Figure 5.11. Let us now examine this molecule and dissect it into its structural components to see if we can understand how these are put together. We will reduce this rather complex, and at first sight bewildering, structure to its simplest representation as a series of motifs. This will help us to understand the structure and see its relationships to other structures. We can immediately discern from Figure 5.31 that the molecule is divided into two clearly separated domains that seem to be of similar size. For the next step we would need a stereopicture of the model or, much better, a graphics display where we could manipulate the model and look at it from different viewpoints. Here instead we have made a schematic diagram of one domain (Figure 5.12), which is normally not done until the analysis is completed and the structural principles are clear. The domain structure has a simple topology We will now follow the main polypeptide chain and trace out a topological diagram for this domain. We can immediately see from Figure 5.13 that the only secondary structure in the molecule is made up of [3 strands, which are arranged in an antiparallel fashion into two separate [3 sheets. Beta strands 1, 2., 4, and 7 form one antiparallel [5 sheet with the strand order 2 l 4 7. We thus draw the left four arrows in Figure 5.13 and connect strands 1 and 2. Similarly, we see that [3 ...
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