Research Projects

HIV-1 Integrase Branched PolymersThe Structural Biology of Retroviral Integrases. Retroviral integrase (IN) catalyzes the incorporation of viral cDNA into the host genome. The design of effective pharmacological treatments remains of paramount importance to the treatment of HIV/AIDS, and detailed structural models of intact IN oligomers in their various forms are essential to new structure-based drug design efforts. My work on the retroviral integrase (IN) has focused on the understanding of higher-order structure and oligomeric forms of the full-length integrase when bound to host factors and DNA, with the overall goal of determining the molecular details of the larger macromolecular assemblies that underlie the steps of retroviral integration and other stages of the viral life cycle. My contributions to-date have greatly informed the field’s understanding of the quaternary structure and stoichiometry of IN, IN-DNA, and IN-host factor assemblies, including experimental confirmation of the quaternary solution structure of the intasome indicated by crystallographic symmetry operations in the case of the prototype foamy virus integrase in complex with DNA. Most recently these approaches have been brought to bear on an exciting new class of allosteric inhibitors (“ALLINIs”) that are able to inhibit IN via selective modulation of its oligomeric properties. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. In 2016, we reported a breakthrough in the structural biology of HIV Integrase: the first crystal structure of HIV-1 Integrase in complex with the ALLINI GSK 1264. To our knowledge, this is the first time that full-length HIV-1 integrase has been crystallized. The structure shows GSK1264 bound to the dimer interface of the catalytic domain and positioned at this interface is a C-terminal domain (CTD) from an adjacent IN dimer. In the crystal lattice, IN forms an open polymer mediated by this interaction. Further studies of a panel of ALLINIs show that HIV escape mutants with reduced sensitivity commonly alter amino acids at or near the inhibitor-mediated interface, and that HIV escape mutations often encode substitutions that reduce multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN through this CTD-catalytic-domain interface.

  1. Eilers G*, Gupta K*, Allen A, Murali H, Sharp R, Hwang Y, Bushman FD, Van Duyne GD. Structure of a Minimal HIV-1 IN-Allosteric Inhibitor Complex at 2.93 Å Resolutions: Routes to Inhibitor Optimization. bioRxiv 2022.06.09.495524 (2022).
  2. Gupta K, Allen A, Giraldo C, Eilers G, Sharp R, Hwang Y, Murali H, Cruz K, Janmey P, Bushman F, Van Duyne GD. Allosteric HIV Integrase Inhibitors Promote Formation of Inactive Branched Polymers via Homomeric Carboxy-Terminal Domain Interactions. Structure. 2021 Mar 4;29(3):213-225.e5. 
  3. Eilers G, Gupta K, Allen A, Zhou J, Hwang Y, Cory MB, Bushman FD, Van Duyne G. Influence of the amino-terminal sequence on the structure and function of HIV integrase. Retrovirology. 2020 Aug 31;17(1):28. 
  4. Gupta K*, Turkki V*, Sherrill-Mix S, Hwang Y, Eilers G, Taylor L, McDanal C, Wang P, Temelkoff D, Nolte RT, Velthuisen E, Jeffrey J, Van Duyne GD, Bushman FD. Structural Basis for Inhibitor-Induced Aggregation of HIV Integrase. PLoS Biol. 2016 Dec;14(12):e1002584. 
  5. Gupta K, Brady T, Dyer BM, Malani N, Hwang Y, Male F, Nolte RT, Wang L, Velthuisen E, Jeffrey J, Van Duyne GD, Bushman FD. Allosteric inhibition of human immunodeficiency virus integrase: late block during viral replication and abnormal multimerization involving specific protein domains. J Biol Chem. 2014 Jul 25;289(30):20477-88. PubMed Central 
  6. Larue R, Gupta K, Wuench C, Shkriabai N, Kessl J, Danhart E, Feng L, Van Duyne GD, Debyser Z, Foster M, Kvaratskhelia M. HIV-1 Integrase C-Terminal Domain Interacts With The Cargo Domain of TNPO3 in vitro. J Biol Chem 287(41):34044-58 (2012).
  7. Gupta K, Curtis J, Krueger S, Hwang Y, Hare S, Cherepanov P, Bushman F, Van Duyne GD. Solution Conformations of Prototype Foamy Virus Integrase and Its Stable Synaptic Complex With U5 viral DNA. Structure 20(11):1918-28 (2012).*featured in Li M and Craigie R, Structure 2012 Nov 7; 20(11):1804-5
  8. Gupta K, Diamond T, Hwang Y, Bushman F, Van Duyne GD. Structural Properties of HIV Integrase-LEDGF Oligomers. J Biol Chem 285(26):20303-20315 (2010).

Phenyalanine HydroxylaseStructural and Biophysical Studies of Phenylalanine Hydroxylase. We have been advancing the understanding of the structure and mechanism of phenylalanine hydroxylase (PAH) since 2013, which underlies the disorder phenylketonuria in collaboration with Eileen Jaffe (Fox Chase Cancer Center). We have determined the first intact crystal structures of rat and human PAH in its resting form, and using solution biophysical methods including SAXS, we have advanced a novel model for the structural transition of the resting-state PAH tetramer to its activated form. Our ongoing work is focused on the dimeric components of the PAH quaternary structure equilibrium and possible small molecules that allosterically activate the enzyme.

  1. Arturo EC, Merkel GW, Hansen MR, Lisowski S, Almeida D, Gupta K, Jaffe EK. Manipulation of a cation-π sandwich reveals conformational flexibility in phenylalanine hydroxylase. Biochimie. 2021 Apr;183:63-77. 
  2. Arturo EC, Gupta K, Hansen MR, Borne E, Jaffe EK. Biophysical characterization of full-length human phenylalanine hydroxylase provides a deeper understanding of its quaternary structure equilibrium. J Biol Chem. 2019 Jun 28;294(26):10131-10145. 
  3. Arturo EC, Gupta K, Héroux A, Stith L, Cross PJ, Parker EJ, Loll PJ, Jaffe EK. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer. Proc Natl Acad Sci U S A. 2016 Mar 1;113(9):2394-9. 

Gemin2-SMNBiophysical and Structural Studies of the Survival of Motor Neurons (SMN) protein. The survival motor neuron (SMN) protein forms the oligomeric core of a large protein assembly that facilitates the biogenesis of spliceosomal snRNPs and other RNPS. In this assembly, the SMN protein binds tightly to Gemin2 and an array of additional proteins in higher eukaryotes. Mutations and deletions in the SMN1 gene have been correlated with spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disorder that affects one in six thousand births. In its most severe forms, patients with the disease rarely live past two years of age, with milder forms of the disease allowing survival into adulthood with only minor motor defects. My ongoing work has been focused on the oligomeric properties and structure of the SMN•Gemin2. In recent work, I contributed biophysical analyses including small-angle scattering constraints towards the NMR determination of Gemin2’s structure in complex with its minimal SMN binding domain. Using SAXS, a dramatic conformational change within Gemin2 was discovered when SMN is bound. I have also performed extensive solution studies of the SMN ‘YG box’ oligomerization domain, where nearly half of the known SMA patient mutations are located. Using analytical centrifugation and light scattering, I have discovered that via its YG box, human SMN exists in a dimer-tetramer-octamer equilibrium in solution. The results of these YG box studies are detailed in Martin et. al. 2012, Gupta et. al. 2015, and Gupta et. al. 2021.

  1. Gupta K, Wen Y, Ninan NS, Raimer AC, Sharp R, Spring AM, Johnson MC, Van Duyne GD, Matera AG. Assembly of higher-order SMN oligomers is essential for metazoan viability and requires an exposed structural motif present in the YG zipper dimer. Accepted, Nucleic Acids Research (2021).
  2. Gray KM, Kaifer KA, Baillat D, Wen Y, Bonacci TR, Ebert AD, Raimer AC, Spring AM, Have ST, Glascock JJ, Gupta K, Van Duyne GD, Emanuele MJ, Lamond AI, Wagner EJ, Lorson CL, Matera AG. Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCFSlmb degron. Mol Biol Cell. 2018 Jan 15;29(2):96-110. 
  3. Gupta K, Martin R, Sharp R, Sarachan KL, Ninan NS, Van Duyne GD. Oligomeric Properties of Survival Motor Neuron·Gemin2 Complexes. J Biol Chem. 2015 Aug 14;290(33):20185-99. 
  4. Martin R, Gupta K, Ninan NS, Perry K, Van Duyne GD. The survival motor neuron protein forms soluble glycine zipper oligomers. Structure. 2012 Nov 7;20(11):1929-39. 
  5. Sarachan KL, Valentine KG, Gupta K, Moorman VR, Gledhill JM Jr, Bernens M, Tommos C, Wand AJ, Van Duyne GD. Solution structure of the core SMN-Gemin2 complex. Biochem J. 2012 Aug 1;445(3):361-70. 

Cre-LoxPStructure and Mechanism of Site-Specific Recombinases. Early in my postdoctoral career in the Van Duyne group, I contributed to the structural and mechanistic study of the tyrosine recombinase Cre from bacteriophage P1 and the large serine integrase TP901-1 from Lactococcus lactis. This class of enzymes catalyze a variety of DNA manipulations, including integration and excision of viral genomes, resolution of multimeric genomes to promote faithful segregation, and control of gene expression. Many tyrosine and serine recombinases are active in mammalian cells and are powerful tools for genomic manipulation and the introduction of transgenes. My contributions to these ongoing studies have included application of X-ray crystallography, small angle scattering, analytical centrifugation, biochemical binding and activity assays, and genetic approaches towards mechanistic insights. These studies provide fundamental and detailed insights into the structure and mechanism of tyrosine and serine recombinases and will guide future efforts to engineer new tools for genomic manipulation.

  1. Li H, Sharp R, Rutherford K, Gupta K, Van Duyne GD. Serine Integrase attP Binding and Specificity. J Mol Biol. 2018 Oct 19;430(21):4401-4418. 
  2. Gupta K, Sharp R, Yuan JB, Li H, Van Duyne GD. Coiled-coil interactions mediate serine integrase directionality. Nucleic Acids Res. 2017 Jul 7;45(12):7339-7353. 
  3. Mandali S, Gupta K, Dawson AR, Van Duyne GD, Johnson RC. Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase. J Bacteriol. 2017 Jun 1;199(11).
  4. Gibb B, Gupta K, Ghosh K, Sharp R, Chen J, and Van Duyne, GD. Requirements for Catalysis in the Cre Recombinase Active Site. Nucleic Acids Research 38(17):5817-32 (2010).
  5. Yuan P, Gupta K, Van Duyne GD. Tetrameric structure of a serine integrase catalytic domain. Structure. 2008 Aug 6;16(8):1275-86. 
  6. Ghosh K, Lau C, Gupta K, Van Duyne GD. Preferential Synapsis of loxP Sites Drives Ordered Strand Exchange in Cre-loxP Site-Specific Recombination. Nature Chemical Biology 1(5):275:282 (2005).

Prostaglandin H2 Synthase-1Structure and Mechanism of Prostaglandin H2 Synthase-1. As a graduate student, I studied the structure and mechanism of prostaglandin H2 synthase-1. This integral membrane protein is the site of action of non-steroidal anti-inflammatory drugs (NSAIDs) and creates a key precursor to the formation of all prostaglandins, which are potent local hormones involved in a variety of physiological processes. I have published a review examining the evolutionary relationship of this mammalian drug target to more primitive orthologs. My primary contribution in this field of study was the determination of several X-ray crystal structures of ovine prostaglandin H2 synthase-1 in complex with different cyclooxygenase inhibitors and alternative heme cofactors. Over the years these contributions have influenced the study and development of cyclooxygenase inhibitors and structure-based studies of mechanism.

  1. Gupta K, Selinsky BS. Bacterial and algal orthologs of prostaglandin H₂ synthase: novel insights into the evolution of an integral membrane protein. Biochim Biophys Acta. 2015 Jan;1848(1 Pt A):83-94. 
  2. Gupta K, Selinsky BS, Loll PJ. 2.0 angstroms structure of prostaglandin H2 synthase-1 reconstituted with a manganese porphyrin cofactor. Acta Crystallogr D Biol Crystallogr. 2006 Feb;62(Pt 2):151-6. 
  3. Gupta K, Kaub CJ, Carey KN, Casillas EG, Selinsky BS, Loll PJ. Manipulation of kinetic profiles in 2-aryl propionic acid cyclooxygenase inhibitors. Bioorg Med Chem Lett. 2004 Feb 9;14(3):667-71. 
  4. Gupta K, Selinsky BS, Kaub CJ, Katz AK, Loll PJ. The 2.0 Å resolution crystal structure of prostaglandin H2 synthase-1: structural insights into an unusual peroxidase. J Mol Biol. 2004 Jan 9;335(2):503-18. 
  5. Selinsky BS*, Gupta K*, Sharkey CT, Loll PJ. Structural Analysis of NSAID binding by Prostaglandin H2 Synthase: Time-Dependent and Time-Independent Inhibitors Elicit Identical Enzyme Conformations. Biochemistry 40(17):5172-5180 (2001). (*=co-first authors)