Research Projects

HIV-1 Integrase Branched Polymers

The Structural Biology of Retroviral Integrases. Retroviral integrases (IN) catalyze the incorporation of viral cDNA into the host genome, and in HIV the enzyme has been established as a target for small molecules, with five FDA-approved drugs now available in the clinic. Over my career, my research has focused on the structure retroviral IN in the context of its higher-order organization, its interaction with endogenous host factors, and more recently, its interaction with novel small molecules. My key contributions in this field include experimental confirmation of the quaternary structure of the intasome in solution, a key assembly in integration, using small-angle X-ray and neutron scattering (SAXS/SANS) and structural insights into the enzyme’s interaction with the host factor LEDGF/p75 and transportin-3 (TNPO3). 

More recently, the focus of our work has shifted to an emerging class of potent HIV antivirals called the allosteric inhibitors of integrase (ALLINIs) that is now in human clinical trials. These drugs target the IN-LEDGF/p75 protein-protein interaction and surprisingly block viral particle formation instead of directly interfering with DNA integration. In a breakthrough discovery in 2016, we determined the first-ever crystal structure of full-length HIV-1 integrase bound to the ALLINI GSK1264. This line of investigation has revealed a novel mechanism for the drug-induced branched polymerization of IN, alongside subsequent insights into drug-induced viral escape mutations, and higher resolution atomic structures.

  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).

Biophysical Studies of Nanoparticle Carriers for Therapeutics. Our group has been pioneering new methods to analyze mRNA- and DNA- containing lipid nanoparticles (LNPs) using solution biophysics. These techniques include analytical ultracentrifugation, multiangle light scattering, and small-angle scattering. In different studies currently in submitted and in review, the group has collaborated with both the Brenner and the Mitchell laboratories at Penn to advance the understanding of structure-function relationships of nucleic acid containing LNP formulations. By employing cutting-edge tools like multiangle light scattering (MALS), multiwavelength analytical ultracentrifugation (MW-AUC), and size- exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS), the lab has established links between the composition and assembly of these LNPs and their biological effects in T- cells and mouse models. These three publications listed here illustrate our prior work in this space of nanoparticle carriers for therapeutics, focused on reverse micelles:

  1. O'Brien ES, Fuglestad B, Lessen HJ, Stetz MA, Lin DW, Marques BS, Gupta, K, Fleming KG, Wand AJ. Membrane Proteins Have Distinct Fast Internal Motion and Residual Conformational Entropy. Angew Chem Int Ed Engl 59(27): 11108-11114 (2020)
  2. Fugelstad B, Gupta K, Wand AJ, Sharp, KA. Water Loading Driven Size, Shape, and Composition of CTAB/hexanol/pentane Reverse Micelles. The Journal of Colloid and Interface Science, Mar 22;540:207-217. doi: 10.1016/j.jcis.2019.01.016. Epub 2019 Jan 6 (2019)
  3. Fuglestad B, Gupta K, Wand AJ, Sharp KA. Experimentally Benchmarked Molecular Dynamics Simulations of Cetyl Trimethylammonium Bromide/Hexanol Reverse Micelles. Langmuir Feb 23;32(7):1674-84 (2016)

Phenyalanine HydroxylaseUnderstanding Allostery in Enzymes that Underlie Inborn Errors of Metabolism. The Gupta group has been advancing the understanding of the structure and mechanism of enzymes that underlie inborn errors of metabolism (IEMs). Our initial studies have focused on phenylalanine hydroxylase (PAH), which underlies the disorder phenylketonuria. With the Jaffe lab at Fox Chase Cancer Center, the group helped determine the first intact crystal structures of rat and human PAH in its resting form, and using solution biophysical methods including SAXS, the group has advanced a novel model for the structural transition of the resting-state PAH tetramer to its activated form. While the study of PAH is informed by many decades of investigation, our group’s work in recent years has had significant impact on the understanding of the mechanism and structure of this enzyme. We have recently extended this program to include other allosterically regulated oligomeric enzymes that under IEMs, including cystathionine β-synthase and porphobilinogen synthase.

  1. 1.  Lee HO, Wang L, Gupta K, Dunbrack RL, Majtan T, Kruger WD. Impact of primary sequence changes on the self-association properties of mammalian cystathionine beta-synthase enzymes. Accepted, Protein Science (2024).
  2. 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. 
  3. 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. 
  4. 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)