Structural Analysis of Human Immunodeficiency Virus Type 1 CRF01_AE Protease in Complex with the Substrate p1-p6

Abstract

The effect of amino acid variability between human immunodeficiency virus type 1 (HIV-1) clades on structure and the emergence of resistance mutations in HIV-1 protease has become an area of significant interest in recent years. We determined the first crystal structure of the HIV-1 CRF01_AE protease in complex with the p1-p6 substrate to a resolution of 2.8 Å. Hydrogen bonding between the flap hinge and the protease core regions shows significant structural rearrangements in CRF01_AE protease compared to the clade B protease structure.Based on its genomic diversity, the human immunodeficiency virus type 1 (HIV-1) has been classified into three groups, M (major), N (nonmajor), and O (other/outlier) (16). Group M has been further defined into nine clades (clades A to D, F to H, and J and K) and a number of subclades and circulating recombinant forms (CRFs). HIV-1 protease is one of the major proteins targeted for anti-HIV drug development. The pol gene, which codes for protease, differs by 10 to 15% between clades (7), and sequence diversity within HIV-1 clades has been an important area of study in recent years due to its possible role in altering resistance pathways within the protease (1, 10). In particular, the HIV-1 CRF01_AE protease acquires nelfinavir resistance via an alternative mutational pathway (1), making the detailed study of non-B proteases strongly warranted.Structural studies of clade B protease have led to the successful development of a number of protease inhibitors (PIs). However, the majority of HIV-1 infection cases in the world result from non-clade B variants, and there is limited evidence that non-clade B variants respond differently to currently available PIs (3, 23). Although a large number of clade B protease structures have been solved over the years, to date, very little structural information is available for non-B HIV proteases. The first non-clade B protease structures for clade F were published recently by Sanches et al. (18), and the crystallization of clade C PI complexes has been reported by Coman et al. (4). We present here the crystal structure of an inactive HIV-1 CRF01_AE protease variant (D25N) in complex with a decameric peptide corresponding to the p1-p6 cleavage site within the Gag and Gag-Pro-Pol polyproteins. CRF01_AE was one of the first CRFs to be identified and is now the predominant HIV-1 variant in Southeast Asia (12). The protease was derived from a Japanese patient isolate and has 10 amino acid substitutions (R14K, K20R, E35D, M36I, R41K, P63L, V64I, H69K, L89M, and I93L) compared to that of clade B (Fig. 1A and B).Crystallization and structure determination. The CRF01_AE protease was expressed and purified as previously described (14). The protein was concentrated to 1.8 mg ml Ϫ1 using a 10-kDa molecular size limit Amicon Ultra-15 centrifugal filter device. The decameric p1-p6 peptide (Arg-Pro-Gly-Asn-PheLeu-Gln-Ser-Arg-Pro; Quality Controlled Biochemicals, Inc., Hopkinton, MA) was solubilized in dimethyl sulfoxide and equilibrated with the protein with a fiv...

Monitoring the autoproteolysis of hiv-1 protease by site-directed spin-labeling and electron paramagnetic resonance spectroscopy

“…Additionally, the possibility for dipolar broadening as a result of doubly-labeled protease has been eliminated by pulsed EPR distance measurements that indicated the distances between the labels exceed 25 Å and generally range from 28 -42 Å depending on protease flap position [17]. These values are in general agreement with molecular dynamic simulations [28] and predictions from known crystal structures [29][30][31]. From these findings, buffers for all EPR experiments are prepared at low ionic strength, typically 2 mM NaOAc with no additional salts.…”

HIV-1 Protease-Substrate Coevolution in Nelfinavir Resistance

“…This effect is even more pronounced in subtype AE, where N88S pulls D30 out of the active site (21). We also showed that D30 not only is key for inhibitor binding but also is essential for recognition of the p1-p6 cleavage site (8,15,22). Consequently, the D30N mutation, which lowers affinity for NFV (23), also likely compromises p1-p6 recognition, leading to coevolution of this cleavage site (15).…”

New approaches to HIV protease inhibitor drug design II: testing the substrate envelope hypothesis to avoid drug resistance and discover robust inhibitors

“…Nevertheless, the impact of mutations outside of the active site also impacts evolution of drug resistance. These include changes in the core of HIV-1 protease either due to natural differences in the HIV clades [47] or compensatory mutations to maintain fitness or can involve changes in substrate sequence. Changes in the beta-barrel core of the protease monomers have been associated with compensatory mutations, such the existence of N88D allowing the simultaneous occurrence of D30N and L90M [48].…”