50 Years of structural immunology

Fifty years ago, the first landmark structures of antibodies heralded the dawn of structural immunology. Momentum then started to build toward understanding how antibodies could recognize the vast universe of potential antigens and how antibody-combining sites could be tailored to engage antigens with high specificity and affinity through recombination of germline genes (V, D, J) and somatic mutation. Equivalent groundbreaking structures in the cellular immune system appeared some 15 to 20 years later and illustrated how processed protein antigens in the form of peptides are presented by MHC molecules to T cell receptors. Structures of antigen receptors in the innate immune system then explained their inherent specificity for particular microbial antigens including lipids, carbohydrates, nucleic acids, small molecules, and specific proteins. These two sides of the immune system act immediately (innate) to particular microbial antigens or evolve (adaptive) to attain high specificity and affinity to a much wider range of antigens. We also include examples of other key receptors in the immune system (cytokine receptors) that regulate immunity and inflammation. Furthermore, these antigen receptors use a limited set of protein folds to accomplish their various immunological roles. The other main players are the antigens themselves. We focus on surface glycoproteins in enveloped viruses including SARS-CoV-2 that enable entry and egress into host cells and are targets for the antibody response. This review covers what we have learned over the past half century about the structural basis of the immune response to microbial pathogens and how that information can be utilized to design vaccines and therapeutics. Fifty years ago, the first landmark structures of antibodies heralded the dawn of structural immunology. Momentum then started to build toward understanding how antibodies could recognize the vast universe of potential antigens and how antibody-combining sites could be tailored to engage antigens with high specificity and affinity through recombination of germline genes (V, D, J) and somatic mutation. Equivalent groundbreaking structures in the cellular immune system appeared some 15 to 20 years later and illustrated how processed protein antigens in the form of peptides are presented by MHC molecules to T cell receptors. Structures of antigen receptors in the innate immune system then explained their inherent specificity for particular microbial antigens including lipids, carbohydrates, nucleic acids, small molecules, and specific proteins. These two sides of the immune system act immediately (innate) to particular microbial antigens or evolve (adaptive) to attain high specificity and affinity to a much wider range of antigens. We also include examples of other key receptors in the immune system (cytokine receptors) that regulate immunity and inflammation. Furthermore, these antigen receptors use a limited set of protein folds to accomplish their various immunological roles. The other main players are the antigens themselves. We focus on surface glycoproteins in enveloped viruses including SARS-CoV-2 that enable entry and egress into host cells and are targets for the antibody response. This review covers what we have learned over the past half century about the structural basis of the immune response to microbial pathogens and how that information can be utilized to design vaccines and therapeutics. As we celebrate 50 years of the Protein Data Bank (PDB), it is fitting to start this review with a reflection on the birth of structural immunology that began with landmark papers on antibody structures published 50 years ago in 1971 (1Sarma V.R. Silverton E.W. Davies D.R. Terry W.D. The three-dimensional structure at 6 Å resolution of a human γG1 immunoglobulin molecule.J. Biol. Chem. 1971; 246: 3753-3759Abstract Full Text PDF PubMed Google Scholar, 2Poljak R.J. Amzel L.M. Avey H.P. Becka L.N. Structure of Fab’ New at 6 Å resolution.Nat. New Biol. 1972; 235: 137-140Crossref PubMed Google Scholar). It was a different time back then when pure proteins were much harder to obtain and structures were equally hard to determine. As structural methods, technologies, and computing improved and recombinant protein expression became possible, the opportunities to tackle previously intractable problems in structural immunology, as well as in structural biology in general, exploded to where we are today with a comprehensive understanding of how microbial pathogens are recognized and countered by the immune system. The PDB played a pivotal role in this whole process by collating and curating the structures that could facilitate structure determination of a macromolecule of choice by molecular replacement. The PDB also enabled mining of the rich arsenal of structural data that allowed general principles for immune recognition to be identified and then harnessed for structure-based design of vaccines and therapeutics. In this review, we provide examples and share our thoughts on how structural biology has shaped our understanding of immune receptors and how they function. The immunoglobulin (Ig) molecule is the major antibody recognition receptor of the humoral immune system. The chemical nature of antibodies, including the different fragments (Fab, Fc) (Fig. 1), the two-chain structure (heavy and light chains), and the antibody Y shape, was first revealed by Gerald Edelman and Rodney Porter in late 1950s and in subsequent papers, for which they received the Nobel prize in Physiology or Medicine in 1972 (see https://www.nobelprize.org/prizes/medicine/1972/porter/lecture/ and https://www.nobelprize.org/prizes/medicine/1972/edelman/lecture/). Porter showed that one of the three antibody fragments that were isolated after papain digestion of rabbit antibodies was able to crystallize, and it was later appropriately named Fc for Fragment crystallizable (3Porter R.R. The hydrolysis of rabbit γ-globulin and antibodies with crystalline papain.Biochem. J. 1959; 73: 119-126Crossref PubMed Google Scholar). The question then was how these heavy and light chains and their substructures were arranged in molecular detail and how they bound antigen. The answers to these questions came in 1971 and 1972 with landmark structures at 6 Å resolution of two human myeloma proteins that could be isolated from serum where they were produced in excess: an intact human IgG1 called Dob (1Sarma V.R. Silverton E.W. Davies D.R. Terry W.D. The three-dimensional structure at 6 Å resolution of a human γG1 immunoglobulin molecule.J. Biol. Chem. 1971; 246: 3753-3759Abstract Full Text PDF PubMed Google Scholar) and the Fab' of human IgG1 New (2Poljak R.J. Amzel L.M. Avey H.P. Becka L.N. Structure of Fab’ New at 6 Å resolution.Nat. New Biol. 1972; 235: 137-140Crossref PubMed Google Scholar) (Fig. 1) from David Davies and Roberto Poljak and colleagues, respectively. This IgG antibody with a deletion in the hinge region was T-shaped and 2-fold symmetric (1Sarma V.R. Silverton E.W. Davies D.R. Terry W.D. The three-dimensional structure at 6 Å resolution of a human γG1 immunoglobulin molecule.J. Biol. Chem. 1971; 246: 3753-3759Abstract Full Text PDF PubMed Google Scholar). A balsa wood model to represent the structure was built (see ref. (1Sarma V.R. Silverton E.W. Davies D.R. Terry W.D. The three-dimensional structure at 6 Å resolution of a human γG1 immunoglobulin molecule.J. Biol. Chem. 1971; 246: 3753-3759Abstract Full Text PDF PubMed Google Scholar) for a photo of the model), and the structure was computationally modeled later in 1977 by fitting of Fab and Fc coordinates into the 6 Å electron density map (4Silverton E.W. Navia M.A. Davies D.R. Three-dimensional structure of an intact human immunoglobulin.Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5140-5144Crossref PubMed Google Scholar). Very few of these early structures were deposited immediately to the PDB; however, many later became available as refined and sometimes higher-resolution structures. Structures of Fab' New with antigen (vitamin K1OH) at 3.5 Å followed shortly thereafter (5Amzel L.M. Poljak R.J. Saul F. Varga J.M. Richards F.F. The three dimensional structure of a combining region-ligand complex of immunoglobulin NEW at 3.5 Å resolution.Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 1427-1430Crossref PubMed Scopus (0) Google Scholar) as well as a higher-resolution structure to 2 Å (PDB ID: 7FAB) (6Poljak R.J. Amzel L.M. Chen B.L. Phizackerley R.P. Saul F. The three-dimensional structure of the Fab' fragment of a human myeloma immunoglobulin at 2.0 Å resolution.Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3440-3444Crossref PubMed Scopus (0) Google Scholar). Structures soon followed for “Bence Jones” immunoglobulin light chain dimers that are excreted into the urine of patients with multiple myelomas and were first discovered and studied by Dr Henry Bence Jones in the late 1840s (7Jones H.B. On a new substance occurring in the urine of a patient with mollities ossium.Philos. Trans. R. Soc. Lond. 1848; 138: 55-62Crossref Google Scholar). Individual Bence Jones proteins, such as Mcg and REI, are code named for the patient from whom they were derived. These early structures included the Bence Jones light chain dimer Mcg at 3.5 Å resolution (8Schiffer M. Girling R.L. Ely K.R. Edmundson A.B. Structure of a lambda-type Bence-Jones protein at 3.5 Å resolution.Biochemistry. 1973; 12: 4620-4631Crossref PubMed Google Scholar) and a dimer of the REI Vκ domains at 2.0 Å (PDB ID: 1REI) (9Epp O. Colman P. Fehlhammer H. Bode W. Schiffer M. Huber R. Palm W. Crystal and molecular structure of a dimer composed of the variable portions of the Bence-Jones protein REI.Eur. J. Biochem. 1974; 45: 513-524Crossref PubMed Scopus (0) Google Scholar). Another IgG structure (Kol) was determined at 4 Å in 1976 and revealed a disordered Fc region, whereas a crystal structure at 3.4 Å of the isolated Fc region showed the carbohydrate acting as a bridge between the widely separated CH2 domains, in contrast to the more closely spaced Ig domains in the Fab (later deposited at 2.9 Å as PDB ID: 1FC1) (10Huber R. Deisenhofer J. Colman P.M. Matsushima M. Palm W. Crystallographic structure studies of an IgG molecule and an Fc fragment.Nature. 1976; 264: 415-420Crossref PubMed Scopus (298) Google Scholar). This structure was particularly important as it gave a structural view of a glycosylated protein that was to prove invaluable a few years later when visualizing and trying to interpret carbohydrates on viral glycoprotein antigens. The mouse Fab McPC603 structure at 3 Å with a small molecule ligand, phosphocholine (PDB ID: 2MCP), was for many years the prototypic example for understanding antibody–antigen recognition, where shape and electrostatic complementarity played key roles in the interaction in the antibody-combining site (11Segal D.M. Padlan E.A. Cohen G.H. Rudikoff S. Potter M. Davies D.R. The three-dimensional structure of a phosphorylcholine-binding mouse immunoglobulin Fab and the nature of the antigen binding site.Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4298-4302Crossref PubMed Scopus (0) Google Scholar) (Fig. 2). The antigen-binding end of the antibody molecule was shown to be located in the variable domain (VH, VL) with its six complementarity-determining region (CDR) loops forming the antibody-combining site for interaction with antigen (Fig. 1). Thus, our initial insights into antibody structure and antigen recognition were fundamentally shaped by these early Fab structures with small molecules (reviewed (12Amzel L.M. Poljak R.J. Three-dimensional structure of immunoglobulins.Ann. Rev. Biochem. 1979; 48: 961-997Crossref PubMed Google Scholar)). What was not clear at that time was how antibodies would interact with larger molecules such as proteins. We had to wait until 1986 and 1987 to get our first glimpses of antibody interactions with lysozyme (Fig. 2), which also was the prototypic antigen for protein crystallization and structure methods development, by the same groups of Poljak and Davies with crystal structures at 2.8-Å (later deposited at 2.5 Å as PDB ID: IFDL) and 2.5-Å (PDB ID: 2HFL) resolutions (13Amit A.G. Mariuzza R.A. Phillips S.E. Poljak R.J. Three-dimensional structure of an antigen-antibody complex at 2.8 Å resolution.Science. 1986; 233: 747-753Crossref PubMed Google Scholar, 14Sheriff S. Silverton E.W. Padlan E.A. Cohen G.H. Smith-Gill S.J. Finzel B.C. Davies D.R. Three-dimensional structure of an antibody-antigen complex.Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8075-8079Crossref PubMed Google Scholar). Structures of antibody complexes with influenza neuraminidase were then determined by Peter Colman and colleagues and deposited later as PDB ID: 1NCA, 1NCB, 1NCC, and 1NCD (15Colman P.M. Laver W.G. Varghese J.N. Baker A.T. Tulloch P.A. Air G.M. Webster R.G. Three-dimensional structure of a complex of antibody with influenza virus neuraminidase.Nature. 1987; 326: 358-363Crossref PubMed Scopus (441) Google Scholar). These structures addressed many of the unresolved issues in the field as to whether the binding sites (epitopes) on the protein antigen were linear (consecutive amino acid stretches) or conformational (composed of multiple segments of amino acids) as most epitopes were found to be. The six CDRs in the light and heavy chains were more involved in interactions with proteins than with small molecules and, consequently, more of the antibody amino acids were involved in contacts with proteins (around 15–20) (16Mariuzza R.A. Phillips S.E. Poljak R.J. The structural basis of antigen-antibody recognition.Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 139-159Crossref PubMed Google Scholar), although not all contacts contributed equally to the binding affinity and can vary from antibody to antibody (17Dall'Acqua W. Goldman E.R. Eisenstein E. Mariuzza R.A. A mutational analysis of the binding of two different proteins to the same antibody.Biochemistry. 1996; 35: 9667-9676Crossref PubMed Scopus (0) Google Scholar). Rather than binding in cavities or grooves as for small molecules, the interacting surfaces for proteins tended to be much larger and more undulating. Another key point of controversy in the 1970s and 1980s was whether the antibody–antigen interactions could be described by a lock-and-key mechanism, where neither the antibody nor the antigen changed conformation, or by induced fit, where the antibody or antigen, or both, molded themselves to the other partner to achieve a better fit. The prevailing view at that time was lock and key over induced fit, although the antibody–neuraminidase structure suggested more of a handshake, where antigen and possibly antibody changed conformation on binding (15Colman P.M. Laver W.G. Varghese J.N. Baker A.T. Tulloch P.A. Air G.M. Webster R.G. Three-dimensional structure of a complex of antibody with influenza virus neuraminidase.Nature. 1987; 326: 358-363Crossref PubMed Scopus (441) Google Scholar). In 1991 to 1992, definitive proof for induced fit as the mechanism for some antibodies came from antibody structures with single-stranded DNA (PDB IDs: 1NBV,1CBV) and a peptide (PDB IDs: 1HIN, 1HIL) (18Herron J.N. He X.M. Ballard D.W. Blier P.R. Pace P.E. Bothwell A.L. Voss Jr., E.W. Edmundson A.B. An autoantibody to single-stranded DNA: Comparison of the three-dimensional structures of the unliganded Fab and a deoxynucleotide-Fab complex.Proteins. 1991; 11: 159-175Crossref PubMed Google Scholar, 19Rini J.M. Schulze-Gahmen U. Wilson I.A. Structural evidence for induced fit as a mechanism for antibody-antigen recognition.Science. 1992; 255: 959-965Crossref PubMed Google Scholar). Thus, it seemed that both lock-and-key and induced fit, or aspects of both, were used, which may in retrospect not be surprising, especially with a diversity system like antibodies. From the accumulation of these pioneering studies on antibodies, many of the burning questions in the field had apparently been addressed on how antibodies, from a genetic and structural viewpoint, are able to recognize the enormous universe of potential antigens. Thus, what seemed to remain unresolved at the time were the fine details on how antibodies recognize specific antigens (20Padlan E.A. Anatomy of the antibody molecule.Mol. Immunol. 1994; 31: 169-217Crossref PubMed Scopus (753) Google Scholar). By the beginning of the 1990s, the difficulty in solving antibody structures had been largely overcome. It may not be that obvious now, but antibody Fab structures were quite challenging to solve in the 1970s and 1980s. When one of the authors (Wilson) entered the antibody field as an Assistant Professor in 1982, he talked to the giants in the field, Roberto Poljak and David Davies, to get a reality check on what was the likelihood of determining a new antibody Fab structure. It was somewhat dismaying to find that only 1 in around 25 antibody Fabs (Poljak) resulted in diffraction-quality crystals that led to subsequent structure determination, although another view (Davies) gave slightly better, but still daunting, odds of 1 in 5 to 1 in 10. Thus, it seemed that focusing on a single antibody or small set of antibodies was probably not the best approach. Thus, although we had initiated work on antibodies to influenza hemagglutinin and myohemerythrin peptides, our laboratory broadened our antibody projects to include HIV-1 gp120 V3 peptides, steroids, and diverse proteins. Crystallization was also a bottleneck even after one had painstakingly obtained Fabs from enzymatic cleavage of IgGs elicited in mice using hybridoma technology against the antigen of choice. Even if crystals were obtained, solving the structures required finding heavy atom derivatives for multiple isomorphous replacement using in-house X-ray sources at room temperature. It turned out that, by refining the antibody IgG cleavage and purification conditions, crystallizing the Fabs fortunately did not turn out to be as much of a problem as anticipated. Advances in antibody crystallization and methods, such as streak seeding and cross seeding (21Stura E.A. Wilson I.A. Analytical and production seeding techniques.Methods. 1990; 1: 38-49Crossref Google Scholar, 22Stura E.A. Wilson I.A. Applications of the streak seeding technique in protein crystallization.J. Cryst. Growth. 1991; 110: 270-282Crossref Google Scholar), helped coax antibody Fabs into forming well-ordered crystals. Thus, new methods for Fab structure determination were needed to now keep up with all of the Fab crystals, other than the usual trial and error methods with multiple isomorphous replacement. Molecular replacement (MR) as pioneered by Michael Rossmann (23Rossmann M.G. Blow D.M. 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Kratky C. Halfon Y. Danz H. Webster P. Bartels K.S. Wittmann H.G. Yonath A. Cryocrystallography of ribosomal particles.Acta Crystallogr. B. 1989; 45: 190-199Crossref PubMed Scopus (97) Google Scholar, 30Hope H. Cryocrystallography of biological macromolecules: A generally applicable method.Acta Crystallogr. B. 1988; 44: 22-26Crossref PubMed Scopus (320) Google Scholar). Since antibody structures were now being determined more frequently, it seemed that most pressing problems had been apparently solved, and David Davies, for example, largely exited from the antibody field to pursue other interests. However, a structure of an intact immunoglobulin with a normal hinge region was elusive as these flexible molecules were hard to crystallize. Structures of intact mouse IgGs appeared in 1995 to 1998 from Alex McPherson’s laboratory (PDB ID: 1IGT, 1IGY) (31Harris L.J. Larson S.B. Hasel K.W. Day J. Greenwood A. McPherson A. 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Crystal structure of a neutralizing human IgG against HIV-1: A template for vaccine design.Science. 2001; 293: 1155-1159Crossref PubMed Scopus (712) Google Scholar) further highlighted the asymmetry in the Y-shaped antibody molecule due to the flexible hinge region connecting the Fab domains to the Fc (Fig. 1). But it was not until human antibodies could be isolated or recombinantly expressed did we realize that structural and functional insights in the antibody field were far from over. Description of many new features, such as extra-long CDR H3 loops, large and small insertions and deletions in the antibody, posttranslational modifications such as tyrosine sulfation and glycosylation, would come from studies of how human antibodies responded to human pathogens, which we will return to in later sections. We were also to find out that antibodies could come in other flavors. Single-Ig domain antibodies, also known as nanobodies, or VHH domains when derived from a heavy chain, are much smaller and can fit into smaller nooks and crevices on antigens (35Muyldermans S. Nanobodies: Natural single-domain antibodies.Annu. Rev. Biochem. 2013; 82: 775-797Crossref PubMed Scopus (918) Google Scholar). The first of these nanobody antibody structures was derived from a heavy-chain-only antibody discovered in camels (36Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa E.B. Bendahman N. Hamers R. Naturally occurring antibodies devoid of light chains.Nature. 1993; 363: 446-448Crossref PubMed Scopus (1854) Google Scholar, 37Muyldermans S. Lauwereys M. Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies.J. Mol. Recognit. 1999; 12: 131-140Crossref PubMed Scopus (0) Google Scholar) and then in other members of the camelid family, such as llamas (36Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa E.B. Bendahman N. Hamers R. Naturally occurring antibodies devoid of light chains.Nature. 1993; 363: 446-448Crossref PubMed Scopus (1854) Google Scholar). The first llama nanobody structure at 1.85 Å in 1996 (PDB ID: 1HCV) showed that it adopted an Ig fold similar to that of a VH in a conventional antibody, but with greater hydrophilicity and with only three CDRs available for antigen binding (38Spinelli S. Frenken L. Bourgeois D. de Ron L. Bos W. Verrips T. Anguille C. Cambillau C. Tegoni M. The crystal structure of a llama heavy chain variable domain.Nat. Struct. Biol. 1996; 3: 752-757Crossref PubMed Scopus (125) Google Scholar) (Fig. 1). These camelid VHH domains were also unusual in that they often contained extra disulfide bonds that could constrain their long CDR H3 loops to CDR H1 (39Muyldermans S. Atarhouch T. Saldanha J. Barbosa J.A.R.G. Hamers R. Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains.Protein Eng. 1994; 7: 1129-1135Crossref PubMed Google Scholar). Another nanobody structure in 1996 in complex with an antigen (PDB ID: 1MEL) showed how its long CDR3 (without an extra disulfide) could penetrate deeply into the active site of lysozyme confirming that these nanobodies could indeed access recessed sites (40Desmyter A. Transue T.R. Ghahroudi M.A. Thi M.H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme.Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (373) Google Scholar) (Fig. 2). The use of only three binding loops compared with six in a conventional antibody did not seem to adversely affect specificity and potency. Cartilaginous fish such as nurse sharks also have heavy-chain-only antibodies termed immunoglobulin new antigen receptors that can also bind with high affinity and specificity to antigens, such as lysozyme (PDB IDs: 1SQ2, 1T6V) (41Stanfield R.L. Dooley H. Flajnik M.F. Wilson I.A. Crystal structure of a shark single-domain antibody V region in complex with lysozyme.Science. 2004; 305: 1770-1773Crossref PubMed Scopus (202) Google Scholar) (Fig. 1). The immunoglobulin new antigen receptor VH domains have a very short CDR 2 and therefore use only two CDRs to attain high-affinity binding. Nanobodies from camelids and sharks as well as engineered human VH domains are now being used extensively as reagents for research, immunodiagnostics (42Salvador J.P. Vilaplana L. Marco M.P. Nanobody: Outstanding features for diagnostic and therapeutic applications.Anal. Bioanal. Chem. 2019; 411: 1703-1713Crossref PubMed Scopus (43) Google Scholar), molecular imaging (43Chakravarty R. Goel S. Cai W. 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Unusual features are also found in antibodies from other animals such as cows, where a subset of cow antibodies have a very long CDR H3 (60 residues or more) encoded primarily by a superlong D region that contains several disulfid