Kalyan Das | Rutgers, The State University of New Jersey (original) (raw)

Papers by Kalyan Das

Nature

Transcription initiation is a key regulatory step in gene expression during which RNA polymerase ... more Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the transition to the elongation phase. Mitochondria recruit a single-subunit RNAP and one or two auxiliary factors to initiate transcription. Previous studies have revealed the molecular architectures of yeast1 and human2 mitochondrial RNAP initiation complexes (ICs). Here we provide a comprehensive, stepwise mechanism of transcription initiation by solving high-resolution cryogenic electron microscopy (cryo-EM) structures of yeast mitochondrial RNAP and the transcription factor Mtf1 catalysing two- to eight-nucleotide RNA synthesis at single-nucleotide addition steps. The growing RNA–DNA is accommodated in the polymerase cleft by template scrunching and non-template reorganization, creating stressed intermediates. During early initiation, non-template strand scrunching and unscrunching destabi...

Antiviral Therapy

HIV-1 reverse transcriptase (RT) genotypes were obtained from 13 patients treated with stavudine.... more HIV-1 reverse transcriptase (RT) genotypes were obtained from 13 patients treated with stavudine. No previously-reported mutations indicative of stavudine resistance were found in these patients and no novel mutations occurred in more than two patients. One patient, treated with stavudine for 1 month and treated previously with zidovudine, zalcitabine and lamivudine, carried a mutation at codon 75 of the RT (V75M). A chimeric virus, including the patient's RT sequence from codon 25 to codon 220, which carried the resistance mutations M41L, D67N, T69D, K70R, L210W and T215Y in addition to V75M, displayed reduced susceptibility to multiple nucleoside RT inhibitors (NRTIs). Removal of V75M from this RT background resulted in a return of susceptibility to didanosine and lamivudine. Our data are in agreement with previous studies demonstrating the rarity of stavudine resistance mutations in stavudine-treated patients. However, we describe a new set of mutations, found in the RT of a ...

Viruses, 2021

In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein pr... more In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs) express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 Å resolution crystal structure of the mature PR-RT from prototype FV (PFV) that can carry out both proteolytic processing and reverse transcription but is in a configuration not competent for proteolytic or polymerase activity. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNa...

HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the vira... more HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3’-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography and identified binding of two to P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized of which two showed noticeable RT inhibition. An engineered RT/DNA aptamer complex trapped the transient P-pocket in solution. Structures of the RT/DNA complex were determined with a fragment and a synthesized analog bound at P-pocket by single-particle cryo-EM. Identification of P-pocket and the devised structure-based platform provide an opportunity for designing new types of ...

STAR Protocols, 2021

In yeast mitochondria, transcription initiation requires assembly of mitochondrial RNA polymerase... more In yeast mitochondria, transcription initiation requires assembly of mitochondrial RNA polymerase and transcription initiation factor MTF1 at the DNA promoter initiation site. This protocol describes the purification of partially melted and fully melted initiation complex states. Both states co-exist in equilibrium in the same sample as seen by cryoelectron microscopy (cryo-EM) and allow elucidation of MTF1's structural roles in controlling the transition into elongation. We further outline how analysis of the complex by light scattering, thermal shift assay, and ultrafiltration assay exhibits reproducible results.

SSRN Electronic Journal, 2020

Proceedings of the National Academy of Sciences, 2019

The initiation phase of HIV reverse transcription has features that are distinct from its elongat... more The initiation phase of HIV reverse transcription has features that are distinct from its elongation phase. The first structure of a reverse transcription initiation complex (RTIC) that trapped the complex after incorporation of one ddCMP nucleotide was published recently [Larsen KP, et al. (2018) Nature 557:118–122]. Here we report a crystal structure of a catalytically active HIV-1 RT/dsRNA complex that mimics the state of the RTIC before the first nucleotide incorporation. The structure reveals that the dsRNA-bound conformation of RT is closer to that of RT bound to a nonnucleoside RT inhibitor (NNRTI) and dsDNA; a hyperextended thumb conformation helps to accommodate the relatively wide dsRNA duplex. The RNA primer 3′ end is positioned 5 Å away from the polymerase site; however, unlike in an NNRTI-bound state in which structural elements of RT restrict the movement of the primer, the primer terminus of dsRNA is not blocked from reaching the active site of RT. The observed struct...

Biochemical pharmacology, Jul 6, 2017

α-Carboxy nucleoside phosphonates (α-CNPs) are modified nucleotides that represent a novel class ... more α-Carboxy nucleoside phosphonates (α-CNPs) are modified nucleotides that represent a novel class of nucleotide-competing reverse transcriptase (RT) inhibitors (NcRTIs). They were designed to act directly against HIV-1 RT without the need for prior activation (phosphorylation). In this respect, they differ from the nucleoside or nucleotide RTIs [N(t)RTIs] that require conversion to their triphosphate forms before being inhibitory to HIV-1 RT. The guanine derivative (G-α-CNP) has now been synthesized and investigated for the first time. The (L)-(+)-enantiomer of G-α-CNP directly and competitively inhibits HIV-1 RT by interacting with the substrate active site of the enzyme. The (D)-(-)-enantiomer proved inactive against HIV-1 RT. In contrast, the (+)- and (-)-enantiomers of G-α-CNP inhibited herpes (i.e. HSV-1, HCMV) DNA polymerases in a non- or uncompetitive manner, strongly indicating interaction of the (L)-(+)- and the (D)-(-)-G-α-CNPs at a location different from the polymerase su...

Antimicrobial agents and chemotherapy, Jun 10, 2017

HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations emerge that confer r... more HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations emerge that confer resistance to nucleoside RT inhibitors (NRTIs) in clinical use. Q151M, and four associated mutations A62V, V75I, F77L, and F116Y were detected in patients failing therapies to dideoxynucleosides (didanosine, ddI; zalcitabine, ddC) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we have systematically determined crystal structures of wild-type RT/dsDNA/dATP (I), wild-type RT/dsDNA/ddATP (II), Q151M RT/dsDNA/dATP (III), Q151Mc RT/dsDNA/dATP (IV), and Q151Mc RT/dsDNA/ddATP (V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3' -endo and 3' -exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at th...

ACS chemical biology, Aug 18, 2016

HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral D... more HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase active site of RT adopts multiple conformational and structural states while performing this task. The states associated are dNTP binding at the N site, catalytic incorporation of a nucleotide, release of a pyrophosphate, and translocation of the primer 3'-end to the P site. Structural characterization of each of these states may help in understanding the molecular mechanisms of drug activity and resistance, and in developing new RT inhibitors. Using a 38-mer DNA template-primer aptamer as the substrate mimic, we crystallized an RT/dsDNA complex that is catalytically active, yet translocation-incompetent in crystals. The ability of RT to perform dNTP binding and incorporation in crystals permitted obtaining a series of structures - (I) RT/DNA (P-site), (II) RT/DNA/AZTTP ternary, (III) RT/AZT-terminated DNA (N-site)...

Organic & biomolecular chemistry, Jan 27, 2016

As α-carboxy nucleoside phosphonates (α-CNPs) have demonstrated a novel mode of action of HIV-1 r... more As α-carboxy nucleoside phosphonates (α-CNPs) have demonstrated a novel mode of action of HIV-1 reverse transcriptase inhibition, structurally related derivatives were synthesized, namely the malonate , the unsaturated and saturated bisphosphonates and , respectively and the amide . These compounds were evaluated for inhibition of HIV-1 reverse transcriptase in cell-free assays. The importance of the α-carboxy phosphonoacetic acid moiety for achieving reverse transcriptase inhibition, without the need for prior phosphorylation, was confirmed. The malonate derivative was less active by two orders of magnitude than the original α-CNPs, while displaying the same pattern of kinetic behavior; interestingly the activity resides in the "L"-enantiomer of , as seen with the earlier series of α-CNPs. A crystal structure with an RT/DNA complex at 2.95 Å resolution revealed the binding of the "L"-enantiomer of , at the polymerase active site with a weaker metal ion chelation...

Acta Crystallographica Section A Foundations of Crystallography, 1996

Protein Science, 1995

Computer modeling studies have been carried out on three nonnucleoside inhibitors complexed with ... more Computer modeling studies have been carried out on three nonnucleoside inhibitors complexed with human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT), using crystal coordinate data from a subset of the protein surrounding the binding pocket region. Results from the minimizations of solvated complexes of 2‐cyclopropyl‐4‐methyl‐5,11‐dihydro‐5H‐dipyrido[3,2‐b :2′,3′‐e][1,4]diazepin‐6‐one (nevirapine), α‐anilino‐2, 6‐dibromophenylacetamide (α‐APA), and 8‐chloro‐tetrahydro‐imidazo(4,5,1‐jk)(1,4)‐benzodiazepin‐2(1H)‐thione (TIBO) show that all three inhibitors maintain a very similar conformational shape, roughly overlay each other in the binding pocket, and appear to function as π‐electron donors to aromatic side‐chain residues surrounding the pocket. However, side‐chain residues adapt to each bound inhibitor in a highly specific manner, closing down around the surface of the drug to make tight van der Waals contacts. Consequently, the results from the calculated minimi...

Current Opinion in Virology, 2013

Structures of RT and its complexes combined with biochemical and clinical data help in illuminati... more Structures of RT and its complexes combined with biochemical and clinical data help in illuminating the molecular mechanisms of different drug-resistance mutations. The NRTI drugs that are used in combinations have different primary mutation sites. RT mutations that confer resistance to one drug can be hypersensitive to another RT drug. Structure of an RT-DNAnevirapine complex revealed how NNRTI binding forbids RT from forming a polymerase competent complex. Collective knowledge about various mechanisms of drug resistance by RT has broader implications for understanding and targeting drug resistance in general. In Part 1, we discussed the role of RT in developing HIV-1 drug resistance, structural and functional states of RT, and the nucleoside/nucleotide analog (NRTI) and non-nucleoside (NNRTI) drugs used in treating HIV-1 infections. In this part, we discuss structural understanding of various mechanisms by which RT confers antiviral drug resistance.

Proceedings of the National Academy of Sciences, 2015

Significance The polymerization of nucleotides by DNA polymerases occurs through a common mechani... more Significance The polymerization of nucleotides by DNA polymerases occurs through a common mechanism based on similar highly conserved amino acid motifs and the universal role of the coordination of Mg 2+ by three dNTP phosphate oxygens. Based on these universal principles, we aimed at designing a dNTP mimic that could interact with a broad variety of DNA polymerases and should consist of three major indispensable entities: a nucleobase for Watson–Crick base-pairing, an enzymatically and chemically stable triphosphate replacement that can efficiently coordinate the Mg 2+ cation, and a variable linker moiety between the nucleobase and the modified phosphate. The resulting α-carboxy nucleoside phosphonates (α-CNPs) were structurally, kinetically, and biochemically investigated, and the novel dNTP mimics were successfully validated in several DNA polymerase models.

Nucleic acids research, 2014

In synthesizing a double-stranded DNA from viral RNA, HIV-1 reverse transcriptase (RT) generates ... more In synthesizing a double-stranded DNA from viral RNA, HIV-1 reverse transcriptase (RT) generates an RNA/DNA intermediate. RT also degrades the RNA strand and synthesizes the second DNA strand. The RNase H active site of RT functions as a nuclease to cleave the RNA strand; however, the structural basis for endonucleolytic cleavage of the RNA strand remains elusive. Here we report crystal structures of RT-RNA/DNA-dATP and RT-RNA/DNA-nevirapine (NVP) ternary complexes at 2.5 and 2.9 Å resolution, respectively. The polymerase region of RT-RNA/DNA-dATP complex resembles DNA/DNA ternary complexes apart from additional interactions of 2'-OH groups of the RNA strand. The conformation and binding of RNA/DNA deviates significantly after the seventh nucleotide versus a DNA/DNA substrate. Binding of NVP slides the RNA/DNA non-uniformly over RT, and the RNA strand moves closer to the RNase H active site. Two additional structures, one containing a gapped RNA and another a bulged RNA, reveal ...

Nature Structural & Molecular Biology, 2010

Human immunodeficiency virus (HIV-1) develops resistance to 3′-azido-2′,3′-deoxythymidine (AZT, z... more Human immunodeficiency virus (HIV-1) develops resistance to 3′-azido-2′,3′-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3′ end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTPand primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision. The development of effective anti-HIV drugs and their use in combination therapies has been estimated to have saved more than 3 million years of life 1. However, drug therapies do not cure HIV infections, and drug therapy is lifelong. Prolonged therapies lead to problems with drug toxicity and drug resistance. Understanding the molecular mechanisms of resistance is important for developing more effective drugs and treatment strategies. Many anti-AIDS drugs

Journal of Virology, 2001

ABSTRACTSuccess in treating hepatitis B virus (HBV) infection with nucleoside analog drugs like l... more ABSTRACTSuccess in treating hepatitis B virus (HBV) infection with nucleoside analog drugs like lamivudine is limited by the emergence of drug-resistant viral strains upon prolonged therapy. The predominant lamivudine resistance mutations in HBV-infected patients are Met552IIe and Met552Val (Met552Ile/Val), frequently in association with a second mutation, Leu528Met. The effects of Leu528Met, Met552Ile, and Met552Val mutations on the binding of HBV polymerase inhibitors and the natural substrate dCTP were evaluated using an in vitro HBV polymerase assay. Susceptibility to lamivudine triphosphate (3TCTP), emtricitabine triphosphate (FTCTP), adefovir diphosphate, penciclovir triphosphate, and lobucavir triphosphate was assessed by determination of inhibition constants (Ki). Recognition of the natural substrate, dCTP, was assessed by determination ofKmvalues. The results from the in vitro studies were as follows: (i) dCTP substrate binding was largely unaffected by the mutations, withK...

Journal of Molecular Biology, 2009

The rapid replication of HIV-1 and the errors made during viral replication, cause the virus to e... more The rapid replication of HIV-1 and the errors made during viral replication, cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly challenging. In the absence of an effective vaccine, drugs are the only useful treatment. Anti-HIV drugs work; so far drug therapy has saved more than three million years of life. Unfortunately, HIV-1 develops resistance to all of the available drugs. Although a number of useful anti-HIV drugs have been approved for use in patients, the problems associated with drug toxicity and the development of resistance means that the search for new drugs is an ongoing process. The three viral enzymes, reverse transcriptase (RT), integrase (IN), and protease (PR) are all good drug targets. Two distinct types of RT inhibitors, both of which block the polymerase activity of RT, have been approved to treat HIV-1 infections, nucleoside analogs (NRTIs) and nonnucleosides (NNRTIs), and there are promising leads for compounds that either block the RNase H activity or block the polymerase in other ways. A better understanding of the structure and function(s) of RT and of the mechanism(s) of inhibition can be used to generate better drugs; in particular drugs that are effective against the current drug-resistant strains of HIV-1. In the absence of an effective vaccine, drugs are the only therapeutic tools that can be used to treat HIV-1 infections. Unfortunately, HIV-1 infections cannot be cured, so that drug therapy, once initiated, must be continued for the life of the patient. This places a special burden on the design of anti-HIV drugs: They need to be relatively nontoxic so that they can be used in longterm therapy. HIV-1 replication is error prone (1) (and references within) and the errors that arise during the viral life cycle, together with the rapid replication of the virus in patients, allows the virus to escape the host's immune system and develop resistance to all of the available drugs (2). The virus evolves sufficiently rapidly that, unless the therapy is well