Proteins with a ring

Proteins with a ring

JOEL JANIN MINIREVIEW Proteins with a ring Proteins come in all sizes and shapes. Those which fold into a ring with a large hole in the middle may ...

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JOEL JANIN

MINIREVIEW

Proteins with a ring Proteins come in all sizes and shapes. Those which fold

into a ring with a large hole in the middle may act as a clamp on DNA, a polysaccharide or another protein. Structure 15 July 1994, 2:571-573

We believe we know so much about proteins - when a new NMR or X-ray structure appears, we should just

have to put it into the right box. Yet, now and then, one pops out which fits in none of our boxes. Then, we realize that terrae incognitae still exist - and we see how essential it is to go on determining structures. An example of a completely new fold is the parallel P-helix found in pectate lyase [1] and Pseudomonas alkaline protease [2], (see [3] for review). Unexpected combinations of established folds are almost equally interesting. Soluble monomeric proteins forming a ring with a hole in the middle undoubtedly belong to this category. Two recent papers describe ring-shaped proteins built by the repetition of a simple motif: a righthanded P-a unit in the porcine ribonuclease inhibitor (RI) analyzed by Kobe and Deisenhofer [4], and a pair of a-helices in the soluble lytic transglycosylase (SLT) from Escherichiacoli studied by Thunnissen et al. [5].

Forming a ring...

The right-handed -a unit is the basic element of any a/Pl tertiary structure. It is present 8 times in triosephosphate isomerase (TIM) and 16 times in RI. Instead of forming a barrel as in TIM, the 16 P-ea units of RI are disposed in an open ring about 70 A wide and 32 A thick, with a large hole in the middle (Fig. 1). The RI sequence was known beforehand to contain 15 homologous stretches of 28-29 residues each. They fold into p3-a units and, as the two short non-homologous segments at both ends also adopt the same fold, there are 16 a-helices and 17 -strands in total. The -strands form a parallel -sheet with very little twist, but the connecting a-helices are wider than the strands and, presumably, this imposes the curvature that gives the protein its striking horseshoe shape. Over 20 % of the residues in RI are leucines. These are regularly disposed at the helix-sheet interface and form leucine-rich repeats. Leucine-rich repeats have been found in a variety of proteins, from gonadotropin receptors to DNA repair enzymes, with up to 41 repeats in one protein. As a full ring can only fit 20-21 repeats, larger numbers may form several connected rings, or the ring may become a lockwasher, with the ends of the ring overlapping.

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Fig. 1. Porcine ribonuclease inhibitor. The horseshoe-like structure is made up of a 17-stranded parallel P-sheet (blue arrows) and 16 connecting ct-helices (red coils). The ring has an outer diameter of 70 A, an inner diameter of about 20 A; the open space between the amino- and carboxy-terminal p-strands is 13 A wide. The 98 leucines and 30 cysteines (out of 456 residues) are shown in balland-stick representation. The cysteines have a free SH group (yellow spheres) in the active form of the protein. They undergo oxidation by'an all-or-none mechanism [16] that must require a major conformational change, since no disulphide bonds can be formed with the SH groups placed the way they are. [This drawing has been prepared with MOLSCRIPT [17] and D Bacon's Raster3D program using atomic coordinates kindly provided by B Kobe and J Deisenhofer (University of Texas Southwestern Medical Center, Dallas).]

SLT is a periplasmic enzyme of 70 kDa that carries out

a lysozyme-like cleavage reaction on the bacterial peptidoglycan. The catalytic site resides on a carboxy-terminal domain comprising about 170 residues, which folds like phage T4 lysozyme [5]. This catalytic domain is stacked on top of a ring formed by 27 a-helices arranged in zigzagging pairs (Fig. 2). The overall shape is like a doughnut, about 80A in diameter and with a large hole inside. The a-helices form a two-layered superhelix much like the ,3-strands do in the ,3-helix of pectate lyase. In pectate lyase, the superhelix is straight, but in SLT it is kinked into a U and the mouth of the U

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Structure 1994, Vol 2 No 7

is closed by the small linker domain comprising helices 23 to 27. The ribonuclease inhibitor fold can also be viewed as a right-handed superhelix made of 13-a units, but such a structure cannot be straight because the widths of a -strand and a-helix are too different.

...and a clamp A protein with a hole in the middle clearly deviates from the globular shape we are used to in soluble proteins. Yet it need not break established rules of protein folding [6]. RI and SLT are close-packed structures with well-defined hydrophobic interiors made of the helix-sheet or helix-helix interface within the superhelix. Even their surface-to-volume ratio is not exceptionally large: the solvent accessible surface area of RI is 18 700 A2, halfway between the expected values for a monomeric globular protein and an oligomeric protein having the same molecular weight as RI (17 000A2 and 19700A 2, respectively, as calculated in [7]). The subunit packing in oligomeric proteins often leaves them with a central hole, a cavity or a channel. In some cases, the hole may have functional relevance. Thus, the pentamer formed by the B-subunits in cholera [8] or pertussis [9] toxins has a central channel through which the catalytic A chain may have to pass in order to penetrate the target cell. The dimeric -subunit'of E. coliDNA polymerase III has a spectacularly large central hole [10]. As the [3-subunit is in charge of processivity - making sure that, once started, polymerization

goes on copying the same DNA template - it is hard to resist the temptation of imagining a double helix threaded through the hole, making the protein into a clamp. The temptation is equally great in the case of the amino-terminal fragment of DNA topoisomerase I studied by Lima et al. [11]. Unlike cholera toxin or the 1-subunit of DNA polymerase, topoisomerase I is a monomer. Its 67 kDa polypeptide chain adopts an elaborate non-repetitive fold with four domains disposed in a ring (Fig. 3). Domain I contains a Rossmann fold, domain II (which is mostly -sheet) an oligomer-binding fold [ 12], and domains III and IV are mostly helical. The hole inside the ring is lined by a-helices of domains III and IV on one side, and on the other side by the large P-sheets of domain II, which are curved into a saddle shape reminiscent of another remarkable DNA-binding protein, the TATA box binding protein [13]. Lima et al. [11] suggest that a movement of domains II and III lets the substrate get into the ring, which is wide enough for double-stranded B-DNA to go through. In common with topoisomerase I and DNA polymerase III SLT's substrate is a polymer, a branched one in the case of the peptidoglycan substrate of SLT. The suggestion that the protein clamps onto the substrate threading through the ring also applies to SLT [5]. Returning now to the porcine ribonuclease inhibitor, biochemical data support the idea that pancreatic ribonuclease A binds inside the horseshoe, but it is marginally too big, and the clamp would have to open

Fig. 2. E. coli soluble lytic transglycosylase. (a) Residues 1-360, in blue, form 22 a-helices arranged in a right-handed superhelix with a kink inthe middle near helix H12. This amino-terminal domain is termed the U domain because of its U-shaped conformation. Residues 361-450, in green, form a linker domain with 5 -helices, and residues 451-618, in yellow, form the catalytic domain that resembles phage T4 lysozyme. (b) The catalytic domain has been removed to show the 80 A diameter ring with a 25-30 A hole formed by the 27 helices. [Figures kindly provided by BW Dijkstra (University of Groningen).]

Proteins with a ring Janin

Fig. 3. E.coli DNA topoisomerase I. (a) The 590-residue amino-terminal fragment folds into four domains with a total of 18 a-helices (A-R) and 14 P-strands (1-14). Domain I contains a Rossmann fold. The ring, approximately 70 A in diameter, is formed by the two large P-sheets in domain IIand by helical domains IIIand IV.(b) The hole in the ring (shown here face-on and from one side) is large enough (27 A) for double-stranded DNA to go through. [Figures kindly provided by A Mondrag6n (Northwestern University, Illinois).]

slightly. If inhibiting ribonuclease A is the only function of RI, the need for a clamp mechanism is certainly not obvious. The bacterial ribonuclease barnase has a natural protein inhibitor called barstar that binds to it as tightly as RI does to the pancreatic enzyme, the dissociation constant being of the order of 10-14 M in both cases. Barstar is a small globular protein, and the mode of association seen in the crystalline bamase-barstar complex [14] is not unlike that of protease inhibitors to trypsin or of lysozyme to antibodies [15]. The horseshoe structure of the active form of RI does not, however, explain the remarkable phenomenon of all-ornone oxidation observed in vitro for its 30 cysteine residues [16], which suggests that this protein with a ring may have more than one trick up its sleeve.

6. 7. 8. 9. 10. 11. 12. 13. 14.

References 1. 2.

3. 4. 5.

Yoder, M.D., Keen, N.T. &Jurnak, F. (1993). New domain motif: the structure of pectate lyase C, a secreted plant virulence factor. Science 260, 1503-1507. Baumann, U., Wu, S., Flaherty, K.M. &McKay, D.B. (1993). Threedimensional structure of the alkaline protease from Pseudomonas aeruginosa a two-domain protein with a calcium binding parallel [ roll motif. EMBO J 12, 3357-3364. Chothia, C. & Murzin, AG. (1993). New folds for allU- proteins. Structure 1, 217-222. Kobe, B. & Deisenhofer, J. (1993). Crystal structures of porcine ribonuclease inhibitor, a protein with leucine-rich repeats. Nature 366, 751-756. Thunnissen, A-M.W.H., et al, &Dijkstra, B.W. (1994). Doughnutshaped structure of a bacterial muramidase revealed by X-ray crystallography. Nature 367, 750-753.

15. 16. 17.

Chothia, C. (1984). Principles that determine the structure of proteins. Annu Rev. Biochem. 54, 537-572. Janin, J., Miller, S. & Chothia, C. (1988). Surface, subunit interfaces and interior of oligomeric proteins. J Mot Biol. 204, 155-164. Sixma, T.K., et al., & Hol, W.GJ. (1991). Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. colt Nature 351, 371-377. Stein, P.E., Boodhoo, A., Armstrong, G.D., Cockle, S.A., Klein, M.H. &Read, RJ. (1994). The crystal structure of pertussis toxin. Structure 2, 45-57. Kong, X.P., Onrust, R., O'Donnell, M &Kuriyan, J. (1992). Threedimensional structure of the P-subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell 6 , 425-437. Lima, C.D., Wang, J.C. & Mondrag6n, A. (1994). Three-dimensional structure of the N-terminal fragment of E. coli DNA topoisomerase I. Nature 367, 139-146. Murzin, AG. & Chothia, C. (1992). Protein architecture: new superfamilies. Curr. Opin. Struct. Biol 2, 895-903. Nikolov,'D.B., et al, & Burley, S.K. (1992). Crystal structure of TFIID TATA-box binding protein. Nature 360, 40-46. Guillet, V., apthom, A, Hartley, R.W. & Mauguen, Y. (1993). Recognition between a bacterial ribonuclease, barnase, and its natural inhibitor, barstar. Structure 1, 165-177. Janin, J. & Chothia, C. (1991). The structure of protein-protein recognition sites. J. Biol. Chem. 265, 16027-16030. Fominaya, J.M. & Hofsteenge, J. (1992). Inactivation of ribonuclease inhibitor by thiol-disulfide exchange. J. Biol. Chem. 267, 24655-24660. Kraulis, PJ. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946-950.

Joel Janin, Laboratoire de Biologie Structurale, UMR 9920 CNRS-Universite Paris-Sud, 91198-Gif-sur-Yvette, France.

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