The possible pathways of self-organization of immunoglobulin domains

Biophysica et Biophysica Acta, 490 (1977) 506-514

© Elsevier/North-HollandBiomedical Press BBA 37562 THE POSSIBLE PATHWAYS OF SELF-ORGANIZATION OF IMMUNOG L O B U L I N DOMAINS

V. P. ZAV'YALOV Department of Biochemistry, Crimean Medical Institute, Simferopol (U.S.S.R.) (Received July 2nd, 1976)

SUMMARY It was proposed that the most probable germ of the fl-structure in globular proteins would be an antiparallel fl-hairpin initiated by the flexibility and/or bend formation of the polypeptide chain between the connected strands. The possible pathways of self-organization of immunoglobulin domains from antiparallel fl-hairpins are computed using well-known kinetic limitations. The most favourable structures corresponding to the greatest number of dehydrated bulky hydrophobic groups are selected. One of the two most favorable structures obtained in such a way coincides with the native tertiary structure of the domains.

INTRODUCTION Immunoglobulins are proteins with specific antibody activity or with structural features closely resembling those of antibodies. The basic multi-chain structure of immunoglobulins consists of two light and two heavy chains linked by interchain disulfide bonds [1-6]. Crystallographic results [7-10] indicate that the light chains are composed of two conformationally independent globular regions (domains [11]) and the heavy chains of the G class of immunoglobulins are composed of four domains. The conformation of domains presents a sandwich-like structure consisting of two fl-pleated sheets. In V domains one of the fl-sheets consists of 4-5 strands [9,10] and another sheet consists of 3-4 strands. In C domains one of fl-sheets consists of 4 strands and another fl-sheet consists of 3 strands. There are 4 antiparallel fl-hairpins in V domains and 3 fl-hairpins in C domains [9]. Thus, immunoglobulin domains present a good model for the investigations of highly fl-structural globular proteins' self-organization. The polypeptide chain of protein cannot form a globule simultaneously. At the first step fluctuating regions of the secondary structure are formed in the unfolded polypeptide chain [12-19]. Their formation is determined by interactions of aminoacid residues adjacent along the chain. The most probable germ or nucleus of the fl-structure will be an antiparallel " "Antiparallel fl-hairpin" refers to two antiparallel fl-strands of the fl-pleated sheet connected by a short length of peptide which can form either different bends or a short segment of helix.

507 /%hairpin initiated by flexibility or/and bend formation of polypeptide chain between the connected strands. In this paper we compute the possible pathways of immunoglobulin domains self-organization from antiparallel fl-hairpins using well-known kinetic limitations. The most favorable structures corresponding to the greatest number of dehydrated bulky hydrophobic groups are selected. If the hypothesis is right, the most favorable structure obtained in such a way must coincide with the native tertiary structure of the domains. RESULTS

Description of the model Immunoglobulin domains present the simplest case of highly fl-structural globular protein. Therefore, it was possible to use the simplest model of polypeptide chain, which is shown in Fig. 1. In the first approximation the side groups of aminoacids were modelled by a cube in which the side chain of alanine may be inserted. The variants of the/%sheets tightly packed in the globular proteins are presented in Fig. IC. It can be seen that according to the model the packing of sheets in parallel to each other in a sandwich-like structure has a most compact form. For the computation we used the free energy of dehydration (--AFdehyd,.) of

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508 bulky hydrophobic groups, the free energy of hydrogen bond formation, when fl-hairpins form a/3-sheet, and the free energy of disulfide bonds. For this purpose we used the free energy of dehydration of strong hydrophobic residues taken into account in correspondence with Tanford's data [20] (lie, Trp: 3 kcal/mol; Tyr: 2.9 kcal/mol; Phe: 2.6 kcal/mol; Leu: 2.4 kcal/mol, Val: 1.7 kcal/mol; Cys: 1.4 kcal/ mol; Met: 1.3 kcal/mol). The free energy of hydrogen bond was assumed: 1.5 kcal/mol. The free energy of disulfide bond was assumed: 66 kcal/mol. We computed only the free energy of dehydration of aminoacid residues which were situated on the surface of a fl-hairpin covered by another fl-hairpin. Other factors affecting the stability of intermediate and final structures were not computed either, in particular, the change of free energy of fragments connecting the fl-hairpins. We computed only a pair interaction of adjacent along the chain fl-hairpins because it is known [18, 19] that (a) the probability of pair interaction of polypeptide chain regions is always far greater than the probability of simultaneous interaction of several regions and (b) the probability of interactions of polypeptide chain regions rapidly decreases with the increase of the number of residues forming the loop between the connected regions. As the conformations of different V and C domains of different chains and species show a similarity [7-10] one can propose the similarity of its self-organization. Therefore, for our investigations we selected VL and CL domains of immunoglobulin G1 (2) New [9] which structure was resolved to 2 A.

The possible pathways of self-organization of VL domain of immunoglobulin G1 (2) New The situation of side chains of aminoacid residues on opposite surfaces of /3-hairpins in VLdomain of immunoglobin G1 (2) New is shown in Fig. 2. It can be seen that one of the surfaces is more hydrophobic than another. Therefore, one can select the surfaces of fl-hairpins which gave the highest free energy of dehydration. The computed pathways of self-organization of the VL domain are presented in Fig. 2. If the letters corresponding to the fl-hairpins are in the same graph, the/3-pleated sheet is formed. If one of the letters is under another, the sandwich-like structure is formed and the covered surfaces of the fl-hairpins are dehydrated. The parallel and antiparallel interpositions of the/%hairpins in a sandwich-like structure are marked by arrows. It can be seen that four of the computed structures are able to form a disulfide bond and therefore have a significantly lower energy than others. Two of the disulfide-bonded structures have lower free.energy than two others. One of the most favorable structures has an overall conformation corresponding to the native structure OfVLdomain. It can be seen that in the native-like structure one of the surfaces of /3-pleated sheet is hydrophobic and another is hydrophilic. In the alternative structure the bulky hydrophobic side groups are located on both surfaces. Thus the native-like structure is more favorable in relation to the quaternary structure formation (association of chains). The "native" pathways of VL domain self-organization are presented in Fig. 3.

The possible pathways of self-organization of CL domain of immunoglobulin G1 (2) New When we computed the possible pathways of CL domain self-organization using three fl-hairpins presented in Fig. 4, the native-like structure was not obtained

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Fig. 2. The possible pathways of self-organization of VL-domain of immunoglobulin (31 (2) New [9]. The location of side chains of aminoacid residues on the opposite surfaces of E-hairpins is presented in the upper part of the figure. The bulky hydrophobic side groups are marked by solid squares. If the letters corresponding to the E-hairpins are in the same graph, the E-sheet is formed. If one of the letters corresponding to the E-hairpins is under another the sandwich-like structure is formed (see Fig. 1C) and the connected surfaces of the/~-hairpins are dehydrated. The parallel and antiparaUel interpositions of the E-structure hairpins in a sandwich-like structure are marked by arrows. For the free energy computation see the text.

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Fig. 4. The possible pathways of self-organization of CL domain of immunoglobulinG1 (2) New [9]. The location of amino acid residues side chains on the opposite surfaces of fl-hairpins is presented in the upper part of the figure. The bulky hydrophobic side chains are marked by solid squares. If the letters corresponding to the fl-hairpins are in the same graph, the fl-sheet is formed. If one of the letters corresponding to the fl-hairpins is under another, the sandwich-like structure is formed (see Fig. 1C) and the connected surfaces of the fl-hairpins are dehydrated. The parallel and antiparallel interposition of the fl-hairpins in a sandwich-like structure is marked by arrows. For the free energy computations see the text. a, a strand of the fl-pleated sheet.

as a result o f such c o m p u t a t i o n . W e analysed the cause of that a n d concluded t h a t the distance between A a n d B fl-hairpins is twice as m u c h as t h a t between B a n d C. But the p r o b a b i l i t y of interactions of polypeptide c h a i n regions rapidly decreases with the increase of the n u m b e r of residues f o r m i n g the loop between the c o n n e c t e d

512 regions. Therefore, the probability of interactions of A and B hairpins with some region of connecting polypeptide chain is higher than the probability of interactions between the hairpins. The region selected was that of AB loop (the length ot polypeptide chain between A and B regions) corresponding to the lowest energy of dehydration as a result of packing into the sandwich-like structure (Fig. 1C) and the possible pathways of self-organization were computed when the "a" region was taken into account. It can be seen from Fig. 4 that the results of computation are very similar to those obtained for the VL domain. Four of the computed structures allow

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Fig. 5. The "native" pathways of CL domain self-organization. The location of side chains of aminoacid residues on opposite surfaces of fl-hairpins is presented in the upper part of the figure. The bulky hydrophobic side chains are marked by the inked squares. For the free energy computations see the text. it, a strand of the fl-plcated sheet.

513 the formation of a disulfide bond and therefore have a significantly lower free energy than others. Two of the disulfide-bonded structures correspond to lower free energy of dehydration than two others. One of the most favorable structures has an overall conformation like the native structure of CL domain. It can be seen that the native-like structure is more favorable in relation to the quaternary structure formation than an alternative structure, because it allows dehydration of more bulky hydrophobic groups. The "native" pathways of CL domain self-organization are presented in Fig. 5. DISCUSSION In the Introduction we proposed that the most probable germ or nucleus of the fl-structure in globular proteins will be an antiparallel fl-hairpin. Using well-known kinetic limitations the possible pathways of self-organization of immunoglobulin domains from antiparallel fl-hairpins were computed. The stability of intermediate and final structures was computed taking into account the free energy of dehydration of bulky hydrophobic groups, the free energy of hydrogen bonds between the peptide groups in the fl-structure and the free energy of disulfide bond formation. From the computation it is found that one of two most favorable structures coincides with the native tertiary structure of the domains. Besides that the native-like structure is more favorable in relation to quaternary structure formation (association of chains) than the alternative structure. The first attempts to obtain theoretically the tertiary structure of globular protein and to predict the possible pathways of its self-organization were made [11, 12, 18, 19] for the simplest case of highly helical proteins of the globin type. We made a similar attempt for another simple case of highly fl-structural proteins of immunoglobulin domain type. The work demonstrates that the "germ hypothesis" [12-19] and well-known kinetic limitations sharply decrease the number of possible conformations of polypeptide chain and facilitate the determination of the native structure of a molecule. Results obtained serve as the working hypothesis in our experimental investigations of self-organization of immunoglobulin domains. ACKNOWLEDGEMENTS The author wishes to thank Professor O. B. Ptitsyn, Dr. V. I. Lim, Dr. A. V. Finkelstein and Dr. A. A. Rashin of the Institute of Protein Research (Poustchino) for helpful discussion of these results.

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