Conformational diversity of gp120 is a central feature of its biological function in entry and immune evasion. In terms of immune evasion, part of the conformational diversity relates to the large rearrangements induced by CD4, allowing for highly conserved surfaces that make up the coreceptor-binding surface to be hidden from the immune system prior to CD4 binding at the cell surface (27
). Part also relates to the decoy strategies involving the elicitation of nonneutralizing antibodies (7
). The bottom line is that gp120 in many ways resembles the conformational machines involved in fusion, rather than the generally more rigid receptor-binding components (38
). While the unusual nature of gp120 has been explored with methods ranging from X-ray crystallography to titration calorimetry and glutaraldehyde fixation of antigenic populations, none of these allow for a spatially resolved portrait of local conformational stability. Here we use HDX to establish such a portrait of HIV-1 gp120, in unliganded and CD4-bound states.
The usefulness of HDX as a probe of local conformational stability is well established (2
), and a number of different HDX methodologies have been developed. Virtually all of these use mass spectroscopy to assess the degree of deuterium incorporation in dismantled fragments, frozen after amide hydrogen/deuterium exchange by low pH and then reassembled into a spatially resolved portrait of the intact protein. We modified established methods to measure local conformational stability (Fig. ). Many of these modifications were previously anticipated (e.g., maximum entropy methods, which extract the maximally unbiased probability distributions of exchange rates from the frequencies of exchange and their standard deviations) (34
). We used previously described software from S. W. Englander and colleagues to calculate the rate constants of the peptic fragments investigated by HDX as theoretically unstructured peptides (see reference 3
). These rate constants were normalized by the HDX data to obtain protection factors, which allow our observations to be placed on an absolute scale (14
). The free energies calculated from protection factors are measures of conformational stability (13
). We observed that NAME-derived energies showed significant correlation with unprocessed exchange frequencies. The average R2
values between NAME energies and unprocessed frequencies were 0.50 for unliganded gp120 and 0.76 for CD4-bound gp120 (see Table S3 in the supplemental material). When mapped onto the crystal structures of unliganded and CD4-bound gp120, HDX frequencies at 50 s generated a picture of conformational stability (see Fig. S4 in the supplemental material), extremely similar to the one produced by the free energies (Fig. ).
We analyzed the correlation between our HDX-determined free energies of local conformational stability and crystal structure-determined parameters of B
value, accessible surface area, and frequencies of hydrogen bonding and β-sheet/α-helix (see Fig. S5 in the supplemental material). Interestingly, the only statistically significant correlations were between CD4-bound gp120 and average accessible surface areas, between CD4-bound gp120 and frequencies of β-sheets/α-helices, or between gp120-bound CD4 and B
value. Our results show that the conformational stabilization of gp120 by CD4 is complex, involving the fixation of regions both distal and proximal to the binding site. This is in contrast to the conformational stabilization of CD4 by gp120, which is localized to the site on CD4 that binds gp120. These observations corroborate crystal structures showing large, global conformational changes in the inner domain of gp120 and small, localized ones in CD4. However, our HDX data suggest that local conformational stability does not predispose a particular region in either gp120 or CD4 to conformational changes but does correspond to larger refolding reactions of domains. For example, crystal structures of SIV unliganded gp120 and HIV-1 CD4-bound gp120 show substantial structural rearrangement in the inner domain but little alteration of the outer domain (6
). Such asymmetry was observed with HDX for both HIV-1 unliganded and CD4-bound gp120: the outer domain was more than an order of magnitude more stable than the inner domain (Fig. ). However, no statistically significant correlation was observed between local changes in structure and changes in conformational stability over the peptic fragments (Fig. ).
Our results indicate that unliganded gp120 is not unstable. How then to explain the extraordinary reduction in entropy in the gp120-CD4 binding reaction if both unliganded gp120 and free CD4 are not unstructured? Theory indicates that reductions in entropy can come from two factors: protein folding or solvent release. Crystallographic studies suggest that at least part of the entropic reduction relates to the release of ordered solvent from the surface of a highly hydrated unliganded gp120 (6
). However, while changes in surface burial correlated well with changes in conformational stability for CD4 (R2
= 0.99), no correlation was observed with gp120, suggesting that solvent exclusion plays a general but complicated role in conformational stability. Likely a combination of solvent effects and the sampling of fewer, more stable, structural conformations together explain observed entropic changes (Fig. ).
FIG. 5. Model of the transition from unliganded to CD4-bound states of gp120. HDX results combined with numerous crystal structures suggest that the unliganded state of HIV-1 gp120 consists of a diverse ensemble of relatively stable conformations, with slow interconversion (more ...)
Recently, analysis of HIV-1 gp120 with intact N and C termini (38
) indicated that a “layered” gp120 architecture, with considerable conformational diversity in the inner domain, may account for a portion of the high entropy content of the unliganded state. The gp120 layers are not unstructured in the manner of a flexible loop but exist as a diverse ensemble of conformations, each significantly different from the other and each with significant conformational stability, all anchored to a conformationally invariant β-sandwich. Binding by CD4 reduces this diverse and highly entropic, though not unstructured, ensemble to a “single” conformation. In line with this, our results show that gp120 fragments spanning the β-sandwich (fragments 1 [R81-N92], 5 [Q114-F223], and 16 [Y486-E492]) display much less change in conformational stability upon CD4 binding than fragments spanning the layers (fragments 2 [N98-E106], 3 [D107-L111], and 4 [Q114-F223]) (Table ). In particular, our data suggest that stabilization of the α-helix spanned by fragment 3 (D107-L111) is important for the conformational convergence in the inner domain. The present HDX analysis of HIV-1 gp120 thus provides not only definition of local conformational flexibility but also insight into the thermodynamic and structural parameters governing the CD4-gp120 binding reaction.