- Email: [email protected]
Blocking HIV-1 gp120 at the Phe43 Cavity: If the Extension Fits…
Structure
Previews Blocking HIV-1 gp120 at the Phe43 Cavity: If the Extension Fits. Barna Dey1 and Edward A. Berger1,* 1Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.05.004
The Phe43 cavity is a mysterious feature in crystallographic structures of HIV-1 gp120-CD4 complexes. In this issue of Structure, Acharya and colleagues provide structural explanations for the potent neutralization by CD4 mimetic miniproteins with chemical extensions that fit into this cavity. The first X-ray crystallographic structures HIV entry inhibitors that act by filling the HIV-1 pseudovirus infection, as much as of HIV-1 gp120, initially solved for the Phe43 cavity were designed either as >1,000-fold for miniprotein M48U1 HXBc2 laboratory-adapted strain (Kwong small organic molecules (Madani et al., against some isolates. et al., 1998) and subsequently refined 2008; Curreli et al., 2012) or as engineered In this issue of Structure, Acharya et al. and extended to the YU2 primary isolate CD4 mimetic ‘‘miniproteins’’, pioneered (2013) analyze the structural basis for (Kwong et al., 2000) (Huang et al., 2004), by Vita et al. (1998) and based on small the extreme neutralization potency of revealed not only tantalizing insights into protein scaffolds. The latter were taken M48U1 as well as the related M48U7, receptor interactions, but also a very puz- through a series of structure-guided opti- which contains a different flexible hydrozling structural feature. In the ternary mizations, involving the inclusion of Phe at phobic extension at the same position. complex of gp120 ‘‘core’’ bound to a position 23 of the miniproteins to mimic Surface plasmon resonance analyses fragment of CD4 and a Fab against a CD4 Phe43 in plugging the cavity (Martin indicated that M48U1, the more potent conserved determinant involved in et al., 2003), and culminated in the addi- CD4 mimetic, bound to gp120 with coreceptor binding, an interfacial pocket tion of flexible hydrophobic extensions extremely high affinity (KD = 0.15 nM), of 150 A˚3 was evident at the nexus to this miniprotein residue, which are which reflected both a high association of three gp120 domains: the inner capable of fitting into the cavity (Van Her- rate and an extremely slow dissociation domain, the outer domain, and the rewege et al., 2008). The result was a dra- rate. Crystallographic analyses of these bridging sheet. The pocket was capped matic increase in neutralization potency of mimetics bound to a gp120 core protein (YU2 isolate) yielded interby Phe43 of CD4, a residue esting insights into their previously shown to be modes of action. Analyses critical for the gp120-CD4 of the flexibilities of the binding interaction. The extended moieties in the structural and functional sigbound structures, coupled nificance of this so-called with the determination of Phe43 cavity (Figure 1) was combined fit parameters unclear, but its conservation (shape complementarity and and unlikely existence in the extent of surface burial), highabsence of CD4 suggested lighted the superiority of that it reflects large recepM48U1 over M48U7 and other tor-induced conformational related miniproteins. The efchanges in gp120. Beyond fects of the miniproteins on its relationship to gp120 the local conformation of function, the Phe43 cavity gp120 were analyzed by comwas recognized as a potenparison of the miniprotein tially useful target for bound with the unliganded antiviral strategies. Thus, structures. The analyses resubstituting a bulky hydrovealed that, when bound phobic tryptophan residue to the extension-containing (gp120 S375W) into the cavmimetics, the Phe43 cavity ity partially stabilizes gp120 Figure 1. The Phe43 Cavity resembled more closely that toward the CD4-bound state CD4 (pink; only domain 1 is shown) binding induces formation of an 150 A˚3 interfacial pocket at the nexus of the gp120 inner domain (pale blue), outer region in the unliganded (Xiang et al., 2002), a feature domain (marine blue), and bridging sheet (purple); the phenyl ring of the (ground state) state comthat was explored for potenPhe43 side chain (red) forms a lid to the cavity and is the only contributing pared to the CD4-bound tial vaccine immunogen apcomponent from CD4. This image was generated using MacPyMOL from Protein Data Bank number 1RZK (Huang et al., 2004). state; earlier mimetics lacking plications (Dey et al., 2007). Structure 21, June 4, 2013 ª2013 Elsevier Ltd All rights reserved 871
Structure
Previews the extensions showed the opposite resemblance. The authors concluded that recognition of the ground state by M48U1 and M48U7 is more favorable energetically, contributing to their higher affinities. More expansive neutralization studies (180 isolate panel) revealed potent activity against all except those from Clade A/E. The presence of His at position 375 of gp120 of these isolates (instead of the canonical Ser) is interpreted to explain this resistance, because the His partially fills the Phe43 pocket, thereby hindering access of the extensions (but not CD4 or the mimetics lacking the extensions). This is consistent with previous findings that resistance to M48U1 involves the substitution of Ser375 with more bulky residues. The results are discussed in terms of a new mechanism of action of CD4 binding site ligands beyond the previously described avidity (multivalent forms) and avoidance of conformational change (e.g., mAb VRC01), namely optimization of the fitting of the hydrophobic extensions within the interfacial Phe43 cavity. These new agents and the structural elucidation of the mechanisms underlying their enhanced anti-HIV potencies promise to guide further design of novel neutralizing agents based on optimal
fitting of extensions into the Phe43 cavity. The recent ex vivo and nonhuman primate studies with M48U1 as a vaginal microbicide to prevent HIV-1 sexual transmission (Dereuddre-Bosquet et al., 2012) are highly promising for the potential antiviral use of these miniproteins. Structureguided design and analysis thus continue to pave the way for the development of new CD4 mimetics that not only cap the Phe43 cavity, but also fit energetically favorable extensions deep within its boundaries.
Dey, B., Pancera, M., Svehla, K., Shu, Y., Xiang, S.H., Vainshtein, J., Li, Y.X., Sodroski, J., Kwong, P.D., Mascola, J.R., and Wyatt, R. (2007). J. Virol. 81, 5579–5593.
ACKNOWLEDGMENTS
Madani, N., Scho¨n, A., Princiotto, A.M., Lalonde, J.M., Courter, J.R., Soeta, T., Ng, D., Wang, L.P., Brower, E.T., Xiang, S.H., et al. (2008). Structure 16, 1689–1701.
Work in the authors’ laboratory is supported in part by the Intramural Program of the NIH, NIAID. REFERENCES Acharya, P., Luongo, T.S., Louder, M.K., McKee, K., Yang, Y., Do Kwon, Y., Mascola, J.R., Kessler, P., Loı¨c, M., and Kwong, P.D. (2013). Structure 21, this issue, 1018–1029. Curreli, F., Choudhury, S., Pyatkin, I., Zagorodnikov, V.P., Bulay, A.K., Altieri, A., Kwon, Y.D., Kwong, P.D., and Debnath, A.K. (2012). J. Med. Chem. 55, 4764–4775. Dereuddre-Bosquet, N., Morellato-Castillo, L., Brouwers, J., Augustijns, P., Bouchemal, K., Ponchel, G., Ramos, O.H.P., Herrera, C., Stefanidou, M., Shattock, R., et al. (2012). PLoS Pathog. 8, e1003071.
Huang, C.C., Venturi, M., Majeed, S., Moore, M.J., Phogat, S., Zhang, M.Y., Dimitrov, D.S., Hendrickson, W.A., Robinson, J., Sodroski, J., et al. (2004). Proc. Natl. Acad. Sci. USA 101, 2706–2711. Kwong, P.D., Wyatt, R., Robinson, J., Sweet, R.W., Sodroski, J., and Hendrickson, W.A. (1998). Nature 393, 648–659. Kwong, P.D., Wyatt, R., Majeed, S., Robinson, J., Sweet, R.W., Sodroski, J., and Hendrickson, W.A. (2000). Structure 8, 1329–1339.
Martin, L., Stricher, F., Misse´, D., Sironi, F., Pugnie`re, M., Barthe, P., Prado-Gotor, R., Freulon, I., Magne, X., Roumestand, C., et al. (2003). Nat. Biotechnol. 21, 71–76. Van Herrewege, Y., Morellato, L., Descours, A., Aerts, L., Michiels, J., Heyndrickx, L., Martin, L., and Vanham, G. (2008). J. Antimicrob. Chemother. 61, 818–826. Vita, C., Vizzavona, J., Drakopoulou, E., Zinn-Justin, S., Gilquin, B., and Me´nez, A. (1998). Biopolymers 47, 93–100. Xiang, S.H., Kwong, P.D., Gupta, R., Rizzuto, C.D., Casper, D.J., Wyatt, R., Wang, L.P., Hendrickson, W.A., Doyle, M.L., and Sodroski, J. (2002). J. Virol. 76, 9888–9899.
HHARI Is One HECT of a RING Christopher W. Davies1 and Chittaranjan Das1,* 1Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.05.003
E3 ubiquitin ligases are the last of an enzyme trio that covalently modifies proteins with ubiquitin, facilitating various cellular functions. In this issue of Structure, Duda and colleagues provide the structural basis for the autoinhibited Ariadne-family of E3-ubiquitin ligases.
Ubiquitination, a covalent attachment of the 76-amino-acid polypeptide ubiquitin (Ub) to lysine residues of target proteins, is an important post-translational modification that controls a wide range of cellular processes. Three enzymatic systems facilitate covalent modification of
proteins by Ub: E1 activating enzymes, E2 conjugating enzymes, and E3 Ub ligases. First, Ub is activated via adenylation by E1 and is linked subsequently to a cysteine residue on the enzyme as a thioester complex. Ub is then transferred to the catalytic cysteine on an E2 via
872 Structure 21, June 4, 2013 ª2013 Elsevier Ltd All rights reserved
transthiolation. The final step of the ubiquitination cascade involves the transfer of Ub from the thioester-linked E2 enzyme (E2Ub) to a Lys residue of the substrate mediated by an E3 ligase. Two distinct mechanisms exist for ligation of Ub to substrates. A homologous to the E6AP
Previews Blocking HIV-1 gp120 at the Phe43 Cavity: If the Extension Fits. Barna Dey1 and Edward A. Berger1,* 1Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.05.004
The Phe43 cavity is a mysterious feature in crystallographic structures of HIV-1 gp120-CD4 complexes. In this issue of Structure, Acharya and colleagues provide structural explanations for the potent neutralization by CD4 mimetic miniproteins with chemical extensions that fit into this cavity. The first X-ray crystallographic structures HIV entry inhibitors that act by filling the HIV-1 pseudovirus infection, as much as of HIV-1 gp120, initially solved for the Phe43 cavity were designed either as >1,000-fold for miniprotein M48U1 HXBc2 laboratory-adapted strain (Kwong small organic molecules (Madani et al., against some isolates. et al., 1998) and subsequently refined 2008; Curreli et al., 2012) or as engineered In this issue of Structure, Acharya et al. and extended to the YU2 primary isolate CD4 mimetic ‘‘miniproteins’’, pioneered (2013) analyze the structural basis for (Kwong et al., 2000) (Huang et al., 2004), by Vita et al. (1998) and based on small the extreme neutralization potency of revealed not only tantalizing insights into protein scaffolds. The latter were taken M48U1 as well as the related M48U7, receptor interactions, but also a very puz- through a series of structure-guided opti- which contains a different flexible hydrozling structural feature. In the ternary mizations, involving the inclusion of Phe at phobic extension at the same position. complex of gp120 ‘‘core’’ bound to a position 23 of the miniproteins to mimic Surface plasmon resonance analyses fragment of CD4 and a Fab against a CD4 Phe43 in plugging the cavity (Martin indicated that M48U1, the more potent conserved determinant involved in et al., 2003), and culminated in the addi- CD4 mimetic, bound to gp120 with coreceptor binding, an interfacial pocket tion of flexible hydrophobic extensions extremely high affinity (KD = 0.15 nM), of 150 A˚3 was evident at the nexus to this miniprotein residue, which are which reflected both a high association of three gp120 domains: the inner capable of fitting into the cavity (Van Her- rate and an extremely slow dissociation domain, the outer domain, and the rewege et al., 2008). The result was a dra- rate. Crystallographic analyses of these bridging sheet. The pocket was capped matic increase in neutralization potency of mimetics bound to a gp120 core protein (YU2 isolate) yielded interby Phe43 of CD4, a residue esting insights into their previously shown to be modes of action. Analyses critical for the gp120-CD4 of the flexibilities of the binding interaction. The extended moieties in the structural and functional sigbound structures, coupled nificance of this so-called with the determination of Phe43 cavity (Figure 1) was combined fit parameters unclear, but its conservation (shape complementarity and and unlikely existence in the extent of surface burial), highabsence of CD4 suggested lighted the superiority of that it reflects large recepM48U1 over M48U7 and other tor-induced conformational related miniproteins. The efchanges in gp120. Beyond fects of the miniproteins on its relationship to gp120 the local conformation of function, the Phe43 cavity gp120 were analyzed by comwas recognized as a potenparison of the miniprotein tially useful target for bound with the unliganded antiviral strategies. Thus, structures. The analyses resubstituting a bulky hydrovealed that, when bound phobic tryptophan residue to the extension-containing (gp120 S375W) into the cavmimetics, the Phe43 cavity ity partially stabilizes gp120 Figure 1. The Phe43 Cavity resembled more closely that toward the CD4-bound state CD4 (pink; only domain 1 is shown) binding induces formation of an 150 A˚3 interfacial pocket at the nexus of the gp120 inner domain (pale blue), outer region in the unliganded (Xiang et al., 2002), a feature domain (marine blue), and bridging sheet (purple); the phenyl ring of the (ground state) state comthat was explored for potenPhe43 side chain (red) forms a lid to the cavity and is the only contributing pared to the CD4-bound tial vaccine immunogen apcomponent from CD4. This image was generated using MacPyMOL from Protein Data Bank number 1RZK (Huang et al., 2004). state; earlier mimetics lacking plications (Dey et al., 2007). Structure 21, June 4, 2013 ª2013 Elsevier Ltd All rights reserved 871
Structure
Previews the extensions showed the opposite resemblance. The authors concluded that recognition of the ground state by M48U1 and M48U7 is more favorable energetically, contributing to their higher affinities. More expansive neutralization studies (180 isolate panel) revealed potent activity against all except those from Clade A/E. The presence of His at position 375 of gp120 of these isolates (instead of the canonical Ser) is interpreted to explain this resistance, because the His partially fills the Phe43 pocket, thereby hindering access of the extensions (but not CD4 or the mimetics lacking the extensions). This is consistent with previous findings that resistance to M48U1 involves the substitution of Ser375 with more bulky residues. The results are discussed in terms of a new mechanism of action of CD4 binding site ligands beyond the previously described avidity (multivalent forms) and avoidance of conformational change (e.g., mAb VRC01), namely optimization of the fitting of the hydrophobic extensions within the interfacial Phe43 cavity. These new agents and the structural elucidation of the mechanisms underlying their enhanced anti-HIV potencies promise to guide further design of novel neutralizing agents based on optimal
fitting of extensions into the Phe43 cavity. The recent ex vivo and nonhuman primate studies with M48U1 as a vaginal microbicide to prevent HIV-1 sexual transmission (Dereuddre-Bosquet et al., 2012) are highly promising for the potential antiviral use of these miniproteins. Structureguided design and analysis thus continue to pave the way for the development of new CD4 mimetics that not only cap the Phe43 cavity, but also fit energetically favorable extensions deep within its boundaries.
Dey, B., Pancera, M., Svehla, K., Shu, Y., Xiang, S.H., Vainshtein, J., Li, Y.X., Sodroski, J., Kwong, P.D., Mascola, J.R., and Wyatt, R. (2007). J. Virol. 81, 5579–5593.
ACKNOWLEDGMENTS
Madani, N., Scho¨n, A., Princiotto, A.M., Lalonde, J.M., Courter, J.R., Soeta, T., Ng, D., Wang, L.P., Brower, E.T., Xiang, S.H., et al. (2008). Structure 16, 1689–1701.
Work in the authors’ laboratory is supported in part by the Intramural Program of the NIH, NIAID. REFERENCES Acharya, P., Luongo, T.S., Louder, M.K., McKee, K., Yang, Y., Do Kwon, Y., Mascola, J.R., Kessler, P., Loı¨c, M., and Kwong, P.D. (2013). Structure 21, this issue, 1018–1029. Curreli, F., Choudhury, S., Pyatkin, I., Zagorodnikov, V.P., Bulay, A.K., Altieri, A., Kwon, Y.D., Kwong, P.D., and Debnath, A.K. (2012). J. Med. Chem. 55, 4764–4775. Dereuddre-Bosquet, N., Morellato-Castillo, L., Brouwers, J., Augustijns, P., Bouchemal, K., Ponchel, G., Ramos, O.H.P., Herrera, C., Stefanidou, M., Shattock, R., et al. (2012). PLoS Pathog. 8, e1003071.
Huang, C.C., Venturi, M., Majeed, S., Moore, M.J., Phogat, S., Zhang, M.Y., Dimitrov, D.S., Hendrickson, W.A., Robinson, J., Sodroski, J., et al. (2004). Proc. Natl. Acad. Sci. USA 101, 2706–2711. Kwong, P.D., Wyatt, R., Robinson, J., Sweet, R.W., Sodroski, J., and Hendrickson, W.A. (1998). Nature 393, 648–659. Kwong, P.D., Wyatt, R., Majeed, S., Robinson, J., Sweet, R.W., Sodroski, J., and Hendrickson, W.A. (2000). Structure 8, 1329–1339.
Martin, L., Stricher, F., Misse´, D., Sironi, F., Pugnie`re, M., Barthe, P., Prado-Gotor, R., Freulon, I., Magne, X., Roumestand, C., et al. (2003). Nat. Biotechnol. 21, 71–76. Van Herrewege, Y., Morellato, L., Descours, A., Aerts, L., Michiels, J., Heyndrickx, L., Martin, L., and Vanham, G. (2008). J. Antimicrob. Chemother. 61, 818–826. Vita, C., Vizzavona, J., Drakopoulou, E., Zinn-Justin, S., Gilquin, B., and Me´nez, A. (1998). Biopolymers 47, 93–100. Xiang, S.H., Kwong, P.D., Gupta, R., Rizzuto, C.D., Casper, D.J., Wyatt, R., Wang, L.P., Hendrickson, W.A., Doyle, M.L., and Sodroski, J. (2002). J. Virol. 76, 9888–9899.
HHARI Is One HECT of a RING Christopher W. Davies1 and Chittaranjan Das1,* 1Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.05.003
E3 ubiquitin ligases are the last of an enzyme trio that covalently modifies proteins with ubiquitin, facilitating various cellular functions. In this issue of Structure, Duda and colleagues provide the structural basis for the autoinhibited Ariadne-family of E3-ubiquitin ligases.
Ubiquitination, a covalent attachment of the 76-amino-acid polypeptide ubiquitin (Ub) to lysine residues of target proteins, is an important post-translational modification that controls a wide range of cellular processes. Three enzymatic systems facilitate covalent modification of
proteins by Ub: E1 activating enzymes, E2 conjugating enzymes, and E3 Ub ligases. First, Ub is activated via adenylation by E1 and is linked subsequently to a cysteine residue on the enzyme as a thioester complex. Ub is then transferred to the catalytic cysteine on an E2 via
872 Structure 21, June 4, 2013 ª2013 Elsevier Ltd All rights reserved
transthiolation. The final step of the ubiquitination cascade involves the transfer of Ub from the thioester-linked E2 enzyme (E2Ub) to a Lys residue of the substrate mediated by an E3 ligase. Two distinct mechanisms exist for ligation of Ub to substrates. A homologous to the E6AP