Structure of PG16 in monoclinic and orthorhombic crystal lattices.
To obtain a structural understanding of the PG9 and PG16 antibodies, we proteolytically processed antibody to Fab, purified it, and screened it for crystallization. Initial robotic screens produced microcrystals of PG9 and PG16. Optimization of the PG16 crystallization by additive screening and microseeding produced rectangular needles with dimensions of up to 0.20 × 0.01 × 0.02 mm (see Fig. S1B in the supplemental material). Diffraction extended to 4.0 Å, and structure solution identified three Fabs per asymmetric unit in a C222 lattice. Two of the Fabs appeared to be reasonably ordered, including the complete complementarity-determining region, but a lack of diffraction beyond 4 Å hindered the refinement required for atomic-level definition of the CDR H3. Optimization of the PG9 crystallization condition did not yield crystals suitable for diffraction.
To obtain improved diffraction, we enzymatically deglycosylated Fabs to remove a single light chain N-linked sugar and screened for crystallization. Robotic crystallizations of deglycosylated PG9 produced no crystals; five conditions with crystals of deglycosylated PG16, however, were found (see Fig. S1C in the supplemental material). Optimization enabled the collection of diffraction data to 2.4 Å, and structure solution identified four Fabs per asymmetric unit in a P21 lattice. Refinement resulted in an R value of 21.1% (Rfree = 26.0%) (Table S1).
All four Fabs showed strong similarity with pairwise superposition yielding Cα RMSDs ranging from 0.16 to 0.33 Å for variable domain and 0.21 to 0.98 Å for the entire Fab. Electron density for residues 3 to 209 of the light chain and residues 1 to 214 of the heavy chain could be seen, but residues 100A to 100J of the heavy chain CDR H3 were disordered in all four molecules in the asymmetric unit (Fig. ).
FIG. 1. Crystal structure of the antigen-binding fragment (Fab) of antibody PG16. Anti-HIV-1 antibodies that effectively neutralize HIV-1 often have unusual structural characteristics. With PG16, an extraordinary CDR H3 forms a separate subdomain, which towers (more ...)
To visualize the entire combining region, we used the 2.4-Å refined monoclinic PG16 structure to bootstrap the refinement of the 4.0-Å orthorhombic crystals. Each of the four independent Fabs from the monoclinic lattice was superimposed onto the three independent Fabs in the orthorhombic lattice and rigid body refined. Then the combined model with lowest Rfree
) was used for phasing. Inspection of the resultant electron density found the combining region in two of the molecules reasonably well ordered. The CDR H3 for one of these molecules proved to be sufficiently well defined to allow the entire CDR H3 to be traced (Fig. ; see also Fig. ). Highly restrained refinement (RMSD on bonds of 0.003 Å) led to an R
value of 25.6% (Rfree
, 31.7%) (see Table S1 in the supplemental material). The electrostatics of the Fab are shown in Fig. and indicate that the Fab is mainly positively charged whereas the CDR H3 is negatively charged.
FIG. 3. CDR H3 flexibility and tyrosine clasp. The head of the CDR H3 appears to have the ability to deform substantially in response to different environments, perhaps enabling it to maneuver into a recessed epitope on HIV-1. In contrast, the handle of the CDR (more ...) CDR H3 of PG16 forms a separate subdomain.
The CDR H3 region of PG16 consists of 28 residues (Kabat numbering) (23
), one of the longest CDR H3 regions observed in a human antibody. Overall the structure resembles an axe, extending above the antibody-variable domains. The handle of the axe is formed by the N-terminal residues (AGGP99
) and the C-terminal residues (YYNY100Q
) of the CDR H3; residues AGG98
extend from strand F of the variable domain and hydrogen bond in antiparallel fashion to YNY100Q
, which extends into strand G of the variable domain (Fig. ). In addition to the hydrogen bonding, the N-terminal amino acids are held in place by the side chains of Tyr100N
, which grip Gly98
in a “tyrosine clasp” (Fig. ), a structural motif which is conserved in all seven of the crystallized forms of PG16.
FIG. 2. Structural and chemical properties of the PG16 CDR H3. One of the longest human CDR H3s ever observed, the PG16 CDR H3 is anionic and secured by a number of hydrogen bonds but lacks the hydrophobic core associated with more stable configurations. (A) (more ...)
Continuing from Pro99 at the heel of the handle, the CDR H3 takes a sharp turn to form the head of the axe (Fig. ). Three residues (IWH100B) outline the bottom of the head with a β-strand, which—after a two-residue turn (DD100D) that forms the blade of the axe—forms four hydrogen bonds to the returning strand (VKY100G). This strand continues for three more residues (YDF100J) to outline the top of the head, before turning (N100K) at the butt of the head and returning (NGY100N) to the heel of the handle. Despite extensive H bonding, the head of the axe is ordered in only two of the seven crystallized lattices, perhaps influenced by lattice packing, which in the monoclinic crystal form does not leave enough room for a fully ordered head (Fig. ). Overall the axe is anionic (Fig. ), and many of its residues are aromatic (Fig. ).
Structural homology of PG16 CDR H3.
Six PDB entries were found to contain structural homologs of the CDR H3. The observed structural similarity could be categorized into three families (Fig. ). One family consisted of three homologs, two from the enolase superfamily and one with unknown function (PDB IDs 2NQL, 3DGB [48
], and 2OZ8). The resemblance of blade and butt of the CDR H3 axe motif could be recognized, while the handle region, which is made up by a twisted β sheet in the CDR H3, was replaced by a loop and a helix in all three proteins (Fig. ). Although the structural resemblance in the blade region was high, due to the structural mismatch of the handle region, the RMSD of aligned residues ranged from 3.47 to 3.71 Å.
FIG. 4. Structural homologs of PG16 CDR H3. Analysis of structural homologs allows insight into the function of the PG16 subdomain in other contexts. Six structural homologs were identified from a database of 16,938 proteins. Based on the structural similarity, (more ...)
In a second family, the CDR H3 was matched to a buried region in two pyrophosphorylase proteins (PDB IDs 1JV1 [44
] and 2YQC [34
]), with an RMSD of 2.16 to 2.26 Å (Fig. ). The blade and twisted handle regions were observed to fit better than the butt region, where an insertion can be found in the two proteins. As opposed to being packed on the protein surface, the structural homolog of the CDR H3 in this second family was sandwiched by secondary structure elements on both sides.
The third family of structural homology had only one member (PDB ID 2ICU), which showed a different structural pattern from that of the other two families. The CDR H3 matched to a core segment surrounded by other structural components of the protein. This segment contains more β-sheet content and showed moderate similarity to the CDR H3 with an RMSD of 3.0 Å (Fig. ). Our results suggest that the axe motif is rare in existing protein structures and that, in the few cases where it is found, it is utilized as a structural building block rather than as an independent domain. Thus, in PG16, the CDR H3 protrudes from the antibody and appears to fold as a semi-independent subdomain, whereas all observed structural homologs were integral parts of other domains. Sequence alignments showed that the CDR H3 contains more aromatic residues than do any of the structural homologs (Fig. ); these aromatic residues, however, were spread throughout the CDR H3 and did not assemble into a core (Fig. ).
Unusual features of PG9/PG16 from sequence analysis.
In addition to the extraordinary CDR H3, the sequences of PG9 and PG16 showed two other unusual features, N-linked glycosylation and extensive affinity maturation, as 20.4% of the Vh gene was altered for both PG9 and PG16 and 15.1 and 21.2% of the Vl gene were altered for PG9 and PG16, respectively (Fig. ). In total, 17.5% of the PG9 and PG16 heavy chains (including V-D-J) and 14.5% and 20.9% of the PG9 and PG16 light chains (V-J), respectively, are affinity matured. The IMGT website (29
) and JOINSOLVER (54
) were used to determine germ line genes. For the D and Jl genes, the genomic precursors were ambiguous as the two servers did not rank the same precursor. Nonetheless, IGHD3-3*01 and IGLJ3*02 were designated the common precursor as proposed by JOINSOLVER and IMGT, respectively. Despite similarity in antibody phenotypes, significant differences were observed between the variable domain sequences of PG9 and PG16 (21.9% divergent) (Fig. ). These differences were mapped onto the structures (Fig. ).
FIG. 5. Affinity maturation and sequence differences between PG9 and PG16. Somatic hypermutation alters residues throughout the variable domains of both PG9 and PG16. (A) Sequence alignments of PG16 and PG9 heavy chain with genomic precursor gene (Vh, D, and (more ...) N-linked glycosylation.
N-linked glycosylation is not present in the genomic V domains (Vh, Vk, and Vl) and is a product of somatic hypermutation. The structure of PG16 revealed that its single N-linked glycan extended off the side of the light chain variable domain (see Fig. S3A in the supplemental material). While the location of the N-linked site was substantially separated from the expected location of the antibody paratope, complex N-linked sugars of the type likely to be present at this site are quite large and could conceivably affect antibody recognition. Electron density, moreover, was observed for only the three protein-proximal sugar residues, suggesting considerable glycan mobility.
To determine the influence of the N-linked glycan on neutralization, we used endoglycosidase H to remove most of the glycan, leaving only the protein-proximal N
-acetylglucosamine. The resultant deglycosylated PG9 and PG16 were tested in both Fab and IgG formats and showed slight isolate-dependent alterations in neutralization. Overall, the presence of the N-linked glycan was not required for neutralization (see Fig. S3B, S4, and S5 and Table S2 in the supplemental material). (Parenthetically, we note that Fab and IgG formats for PG9 and PG16 had comparable neutralization potencies [Fig. S3B, S4, and S5 and Table S2]. Related observations have been made elsewhere for CD4-binding-site antibodies [9
] and for select broadly neutralizing ones [24
Domain swapping in structure-function analysis.
Structure-function analysis by hypothesis-driven mutational dissection is a well-established paradigm. With the PG9 and PG16 antibodies, however, two features of interest, affinity maturation and paratope definition, did not appear particularly suited to such a hypothesis-based approach. Affinity maturation affects about a quarter of the residues throughout the variable domains of both PG9 and PG16 (Fig. ), making it difficult to focus on any particular change in altering neutralization; moreover, the solved structure contained only free antibody, not the antibody-epitope complex, making it difficult to focus on a particular site of interaction. We therefore chose to use a resolution-enhancing approach as opposed to a hypothesis-driven approach (39
). We created coarse screens of functional relevance by utilizing chimeric antibodies to take advantage of both the functional similarity and amino acid divergence of the somatically related PG9 and PG16 antibodies. Because the expression of heavy and light chains already involved separate plasmids, chimeras between heavy and light chain variants were made by merely transfecting combinations of different heavy and light chain plasmids during transient-transfection expression. As the CDR H3 comprised almost half of the surface area of the combining region, we chose to segregate its contribution from the rest of the heavy chain and the light chain. Here we describe structure-function analysis of the resolution-enhancing chimeric antibody constructs.
V-gene genomic reversion and neutralization assessment.
To parse the contribution of affinity maturation to neutralization, we reverted the Vl portion of the light chain, altering 15 and 21 residues for PG9 and PG16, respectively, and the Vh portion of the heavy chain, changing 20 residues for both PG9 and PG16. Chimeric antibodies of all six possible combinations (Fig. ) were expressed and tested for neutralization along with wild-type PG9 and PG16 against 18 HIV-1 isolates and two non-HIV-1 virus controls (Fig. ; see also Fig. S6 and Table S3 in the supplemental material).
Overall, isolate-specific results were obtained, indicating that the chimeric V-gene reversions interacted with the HIV-1 Env in different ways. Heavy chain V-gene reversion resulted in chimeric antibodies that were still broad and potent neutralizers, whereas light chain V-gene reversion produced chimeric antibodies that were less able to neutralize. Reversions of both heavy and light V genes were generally inactive, although the fully V-gene-reverted PG9 showed an IC50 of ~5 μg/ml against the clade C isolate ZM233.6 (see Table S3 in the supplemental material). Since expression of the V-gene reverted chimeric antibodies was similar to that of the mature antibodies and they neutralized at least one isolate, we believe that the chimeric antibodies were able to form functional IgG. Despite substantial isolate-specific variation, affinity maturation of PG9 and PG16 correlated with antibody neutralization breadth (P = 0.037) and potency (P < 0.0001) (Fig. ).
Dissection of PG9/PG16 functional differences with antibody chimeras.
Most viruses are neutralized by PG9 and PG16 with less than 10-fold difference in antibody IC50
; some viruses, however, show greater discrimination, being substantially more sensitive to PG9 or to PG16 (58
). To decipher the source of functional differences between PG9 and PG16, swaps of light chain, heavy chain, and CDR H3 were assessed for functional competence, and a full complement of chimeric PG9/PG16 antibodies was tested for neutralization against a panel of HIV-1 isolates (Fig. ; see also Table S4 in the supplemental material).
FIG. 7. PG9 and PG16 swaps and neutralization assessment. (A) PG16- and PG9-swap chimeras were made as shown: PG9 and PG16 light chains were swapped, PG9 and PG16 heavy chains without the CDR H3 changed were swapped, and PG9 and PG16 CDR H3 were swapped. PG16 (more ...)
The PG9/PG16 chimeras were first screened for activity against three pseudoviruses of high (and similar) sensitivities to neutralization by PG9 and PG16. All of these chimeras were functional, and IC50 were within ~100-fold of each other (Fig. ), indicating functional complementation between heavy-light and CDR H3 regions of PG9 and PG16. We next tested pseudoviruses that were preferentially sensitive to either PG9 or PG16 (see Fig. S7 and S8 and Table S4 in the supplemental material) to decipher which parts of the antibody contributed to functional enhancement of each of the antibodies relative to the other. For PG16-sensitive isolates, the PG16 CDR H3 appeared to be the primary contributor; for PG9-sensitive isolates, the PG9 CDR H3 was a dominant factor, although the PG9 heavy chain also had some contribution (Fig. ).
Identification of a common paratope.
Neutralization by PG9 correlates strongly with that of PG16 (58
), indicating that these antibodies recognize a common HIV-1 epitope. This suggests that a common surface on PG9 and PG16 might be involved in recognition of HIV-1. Substantial differences in sequence are found between PG9 and PG16. Mapping of these differences onto the structure of PG16 showed that they were located throughout the variable region (Fig. ). In light of the number and ubiquity of the differences between PG9 and PG16, it is remarkable that their virus neutralization properties are so similar. That is, 34% of the combining region differs between the two antibodies—leaving no completely conserved surface of sufficient size for antigen binding (Fig. ). We therefore asked whether such differences might constrain the potential location of common surface used by PG9 and PG16 to recognize HIV-1. In particular, we asked whether an area of the size of a typical paratope (e.g., roughly 20 × 20 Å) could be found within the combining surface of PG16 that contained conservative amino acid substitutions with PG9 (Fig. ). Because the CDR H3 was the source of functional enhancements, nonconservative substitutions there were allowed. With the rest of the combining surface, nonconservative changes in the heavy chain and the light chain CDR L3 were observed. A potential patch, which was composed of elements from the CDR L1 and L2 and the CDR H3, appeared to be a likely site of HIV-1 Env interactions (Fig. ; see also Fig. S9 in the supplemental material).
FIG. 8. Identification of a potential site on PG16 for recognition of gp120. A combination of structure analysis and resolution-enhancing chimeras permits the boundaries of the likely paratope of PG16 to be identified. (A) Conservative (green) and nonconservative (more ...)