Deepak T. Nair

Replication by human DNA polymerase-|[iota]| occurs by Hoogsteen base-pairing

Almost all DNA polymerases show a strong preference for incorporating the nucleotide that forms the correct Watson–Crick base pair with the template base. In addition, the catalytic efficiencies with which any given polymerase forms the four possible correct base pairs are roughly the same. Human DNA polymerase-ι (hPolι), a member of the Y family of DNA polymerases, is an exception to these rules. hPolι incorporates the correct nucleotide opposite a template adenine with a several hundred to several thousand fold greater efficiency than it incorporates the correct nucleotide opposite a template thymine, whereas its efficiency for correct nucleotide incorporation opposite a template guanine or cytosine is intermediate between these two extremes. Here we present the crystal structure of hPolι bound to a template primer and an incoming nucleotide. The structure reveals a polymerase that is ‘specialized’ for Hoogsteen base-pairing, whereby the templating base is driven to the syn conformation. Hoogsteen base-pairing offers a basis for the varied efficiencies and fidelities of hPolι opposite different template bases, and it provides an elegant mechanism for promoting replication through minor-groove purine adducts that interfere with replication.

Fast native-SAD phasing for routine macromolecular structure determination

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

Epitope Recognition by Diverse Antibodies Suggests Conformational Convergence in an Antibody Response

Crystal structures of distinct mAbs that recognize a common epitope of a peptide Ag have been determined and analyzed in the unbound and bound forms. These Abs display dissimilar binding site structures in the absence of the Ag. The dissimilarity is primarily expressed in the conformations of complementarity-determining region H3, which is responsible for defining the epitope specificity. Interestingly, however, the three Abs exhibit similar complementarity-determining region conformations in the Ag binding site while recognizing the common epitope, indicating that different pathways of binding are used for Ag recognition. The epitope also exhibits conformational similarity when bound to each of these Abs, although the peptide Ag was otherwise flexible. The observed conformational convergence in the epitope and the Ag binding site was facilitated by the plasticity in the nature of interactions.

Crystal Structure of an Antibody Bound to an Immunodominant Peptide Epitope: Novel Features in Peptide-Antibody Recognition

The crystal structure of Fab of an Ab PC283 complexed with its corresponding peptide Ag, PS1 (HQLDPAFGANSTNPD), derived from the hepatitis B virus surface Ag was determined. The PS1 stretch Gln2P to Phe7P is present in the Ag binding site of the Ab, while the next three residues of the peptide are raised above the binding groove. The residues Ser11P, Thr12P, and Asn13P then loop back onto the Ag-binding site of the Ab. The last two residues, Pro14P and Asp15P, extend outside the binding site without forming any contacts with the Ab. The PC283-PS1 complex is among the few examples where the light chain complementarity-determining regions show more interactions than the heavy chain complementarity-determining regions, and a distal framework residue is involved in Ag binding. As seen from the crystal structure, most of the contacts between peptide and Ab are through the five residues, Leu3-Asp4-Pro5-Ala6-Phe7, of PS1. The paratope is predominantly hydrophobic with aromatic residues lining the binding pocket, although a salt bridge also contributes to stabilizing the Ag-Ab interaction. The molecular surface area buried upon PS1 binding is 756 Å2 for the peptide and 625 Å2 for the Fab, which is higher than what has been seen to date for Ab-peptide complexes. A comparison between PC283 structure and a homology model of its germline ancestor suggests that paratope optimization for PS1 occurs by improving both charge and shape complementarity.

Mimicry of native peptide antigens by the corresponding retro-inverso analogs is dependent on their intrinsic structure and interaction propensities.

Retro-inverso (ri) analogs of model T cell and B cell epitopes were predictively designed as mimics and then assayed for activity to understand the basis of functional ri-antigenic peptide mimicry. ri versions of two MHC class I binding peptide epitopes, one from a vesicular stomatitis virus glycoprotein (VSVp) and another from OVA (OVAp), exhibit structural as well as functional mimicry of their native counterparts. The two ri peptides exhibit conformational plasticity and they bind to MHC class I (H-2Kb) similar to their native counterparts both in silico and in vivo. In fact, ri-OVAp is also presented to an OVAp-specific T cell line in a mode similar to native OVAp. In contrast, the ri version of an immunodominant B cell peptide epitope from a hepatitis B virus protein, PS1, exhibits no structural or functional correlation with its native counterpart. PS1 and its ri analog do not exhibit similar conformational propensities. PS1 is less flexible relative to its ri version. These observed structure-function relationships of the ri-peptide epitopes are consistent with the differences in recognition properties between peptide-MHC vs peptide-Ab binding where, while the recognition of the epitope by MHC is pattern based, the exquisitely specific recognition of Ag by Ab arises from the high complementarity between the Ag and the binding site of the Ab. It is evident that the correlation of conformational and interaction propensities of native l-peptides and their ri counterparts depends both on their inherent structural properties and on their mode of recognition.

RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state

Flaviviral RNA-dependent RNA polymerases (RdRps) initiate replication of the single-stranded RNA genome in the absence of a primer. The template sequence 5′-CU-3′ at the 3′-end of the flaviviral genome is highly conserved. Surprisingly, flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. We show that GTP stimulates de novo RNA synthesis by RdRp from Japanese encephalitis virus (jRdRp) also. Crystal structures of jRdRp complexed with GTP and ATP provide a basis for specific recognition of GTP. Comparison of the jRdRpGTP structure with other viral RdRp-GTP structures shows that GTP binds jRdRp in a novel conformation. Apo-jRdRp structure suggests that the conserved motif F of jRdRp occupies multiple conformations in absence of GTP. Motif F becomes ordered on GTP binding and occludes the nucleotide triphosphate entry tunnel. Mutational analysis of key residues that interact with GTP evinces that the jRdRpGTP structure represents a novel pre-initiation state. Also, binding studies show that GTP binding reduces affinity of RdRp for RNA, but the presence of the catalytic Mn2+ ion abolishes this inhibition. Collectively, these observations suggest that the observed pre-initiation state may serve as a checkpoint to prevent erroneous template-independent RNA synthesis by jRdRp during initiation.

The C-Terminal Domain of the MutL Homolog from Neisseria gonorrhoeae Forms an Inverted Homodimer

The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.

A strategically located serine residue is critical for the mutator activity of DNA polymerase IV from Escherichia coli.

The Y-family DNA polymerase IV or PolIV (Escherichia coli) is the founding member of the DinB family and is known to play an important role in stress-induced mutagenesis. We have determined four crystal structures of this enzyme in its pre-catalytic state in complex with substrate DNA presenting the four possible template nucleotides that are paired with the corresponding incoming nucleotide triphosphates. In all four structures, the Ser42 residue in the active site forms interactions with the base moieties of the incipient Watson–Crick base pair. This residue is located close to the centre of the nascent base pair towards the minor groove. In vitro and in vivo assays show that the fidelity of the PolIV enzyme increases drastically when this Ser residue was mutated to Ala. In addition, the structure of PolIV with the mismatch A:C in the active site shows that the Ser42 residue plays an important role in stabilizing dCTP in a conformation compatible with catalysis. Overall, the structural, biochemical and functional data presented here show that the Ser42 residue is present at a strategic location to stabilize mismatches in the PolIV active site, and thus facilitate the appearance of transition and transversion mutations.

Replication across template T/U by human DNA polymerase-ι

Human DNA polymerase-iota (Poliota) incorporates correct nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines. In fact, the fidelity opposite template T is so poor that Poliota inserts an incorrect dGTP approximately 10 times better than it inserts the correct dATP. We determine here how a template T/U is accommodated in the Poliota active site and why a G is incorporated more efficiently than an A. We show that in the absence of incoming dATP or dGTP (binary complex), template T/U exists in both syn and anti conformations, but in the presence of dATP or dGTP (ternary complexes), template T/U is predominantly in the anti conformation. We also show that dATP and dGTP insert differently opposite template T/U, and that the basis of selection of dGTP over dATP is a hydrogen bond between the N2 amino group of dGTP and Gln59 of Poliota.

MsDpo4—a DinB Homolog from Mycobacterium smegmatis—Is an Error-Prone DNA Polymerase That Can Promote G:T and T:G Mismatches

Error-prone DNA synthesis in prokaryotes imparts plasticity to the genome to allow for evolution in unfavorable environmental conditions, and this phenomenon is termed adaptive mutagenesis. At a molecular level, adaptive mutagenesis is mediated by upregulating the expression of specialized error-prone DNA polymerases that generally belong to the Y-family, such as the polypeptide product of the dinB gene in case of E. coli. However, unlike E. coli, it has been seen that expression of the homologs of dinB in Mycobacterium tuberculosis are not upregulated under conditions of stress. These studies suggest that DinB homologs in Mycobacteria might not be able to promote mismatches and participate in adaptive mutagenesis. We show that a representative homolog from Mycobacterium smegmatis (MsDpo4) can carry out template-dependent nucleotide incorporation and therefore is a DNA polymerase. In addition, it is seen that MsDpo4 is also capable of misincorporation with a significant ability to promote G:T and T:G mismatches. The frequency of misincorporation for these two mismatches is similar to that exhibited by archaeal and prokaryotic homologs. Overall, our data show that MsDpo4 has the capacity to facilitate transition mutations and can potentially impart plasticity to the genome.