The remarkable selectivity of N-methyl mesoporphyrin IX (NMM) for G-quadruplexes (GQs) is long known, however its ability to stabilize and bind GQs has not been investigated in detail. Through the use of circular dichroism, UV-visible spectroscopy and fluorescence resonance energy transfer (FRET) melting assay we have shown that NMM stabilizes human telomeric DNA dAG3(TTAG3)3 (Tel22) and is selective for its parallel conformation to which it binds in 1:1 stoichiometry with a binding constant of ∼1.0 × 105 M−1. NMM does not interact with an antiparallel conformation of Tel22 in sodium buffer and is the second example in the literature, after TOxaPy, of a ligand with an excellent selectivity for a specific GQ structure. NMM's stabilizing ability toward predominantly parallel GQ conformation is universal: it stabilizes a variety of biologically relevant G-rich sequences including telomeres and oncogene promoters. The N-methyl group is integral for selectivity and stabilization, as the unmethylated analogue, mesoporphyrin IX, does not stabilize GQ DNA in FRET melting assays. Finally, NMM induces the isomerization of Tel22 into a structure with increased parallel component in K+ but not in Na+ buffer. The ability of NMM to cause structural rearrangement and efficient stabilization of Tel22 may bear biological significance.

N-methyl mesoporphyrin IX (NMM) is exceptionally selective for G-quadruplexes (GQ) relative to duplex DNA and, as such, has found a wide range of applications in biology and chemistry. In addition, NMM is selective for parallel versus antiparallel GQ folds, as was recently demonstrated in our laboratory. Here, we present the X-ray crystal structure of a complex between NMM and human telomeric DNA dAGGG(TTAGGG)(3), Tel22, determined in two space groups, P2(1)2(1)2 and P6, at 1.65 and 2.15 Å resolution, respectively. The former is the highest resolution structure of the human telomeric GQ DNA reported to date. The biological unit contains a Tel22 dimer of 5'-5' stacked parallel-stranded quadruplexes capped on both ends with NMM, supporting the spectroscopically determined 1:1 stoichiometry. NMM is capable of adjusting its macrocycle geometry to closely match that of the terminal G-tetrad required for efficient π-π stacking. The out-of-plane N-methyl group of NMM fits perfectly into the center of the parallel GQ core where it aligns with potassium ions. In contrast, the interaction of the N-methyl group with duplex DNA or antiparallel GQ would lead to steric clashes that prevent NMM from binding to these structures, thus explaining its unique selectivity. On the basis of the biochemical data, binding of NMM to Tel22 does not rely on relatively nonspecific electrostatic interactions, which characterize most canonical GQ ligands, but rather it is hydrophobic in nature. The structural features observed in the NMM-Tel22 complex described here will serve as guidelines for developing new quadruplex ligands that have excellent affinity and precisely defined selectivity.

Nramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward- to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species. Our results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms.

Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA–stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3′ overhang inhibits 5′→3′ resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo.

SUMMARY Clustered protocadherin (Pcdh) proteins mediate dendritic self-avoidance in neurons via specific homophilic interactions in their extracellular cadherin (EC) domains. We determined crystal structures of EC1-EC3, containing the homophilic specificity-determining region, of two mouse clustered Pcdh isoforms (PcdhγA1 and PcdhγC3) to investigate the nature of the homophilic interaction. Within the crystal lattices, we observe antiparallel interfaces consistent with a role in trans cell-cell contact. Antiparallel dimerization is supported by evolutionary correlations. Two interfaces, located primarily on EC2-EC3, involve distinctive clustered Pcdh structure and sequence motifs, lack predicted glycosylation sites, and contain residues highly conserved in orthologs but not paralogs, pointing towards their biological significance as homophilic interaction interfaces. These two interfaces are similar yet distinct, reflecting a possible difference in interaction architecture between clustered Pcdh subfamilies. These structures initiate a molecular understanding of clustered Pcdh assemblies that are required to produce functional neuronal networks.

Guanine quadruplexes (GQ) are four‐stranded DNA structures formed by guanine‐rich DNA sequences. The formation of GQs inhibits cancer cell growth, although the detection of GQs in vivo has proven difficult, in part because of their structural diversity. The development of GQ‐selective fluorescent reporters would enhance our ability to quantify the number and location of GQs, ultimately advancing biological studies of quadruplex relevance and function. N‐methylmesoporphyrin IX (NMM) interacts selectively with parallel‐stranded GQs; in addition, its fluorescence is sensitive to the presence of DNA, making this ligand a possible candidate for a quadruplex probe. In the present study, we investigated the effect of DNA secondary structure on NMM fluorescence. We found that NMM fluorescence increases by about 60‐fold in the presence of parallel‐stranded GQs and by about 40‐fold in the presence of hybrid GQs. Antiparallel GQs lead to lower than 10‐fold increases in NMM fluorescence. Single‐stranded DNA, duplex, or i‐motif, induce no change in NMM fluorescence. We conclude that NMM shows promise as a ‘turn‐on’ fluorescent probe for detecting quadruplex structures, as well as for differentiating them on the basis of strand orientation.

No abstract

13Nramp family transporters-expressed in organisms from bacteria to humans-enable uptake of 14 essential divalent transition metals via an alternating-access mechanism that includes proton co-15 transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp at 16 complementary stages of its transport cycle to provide a thorough description of the Nramp 17 transport cycle by identifying the key intramolecular rearrangements and changes to the metal 18 coordination sphere. Strikingly, while metal transport requires cycling from outward-to inward-19 open states, efficient proton transport still occurs in outward-locked (but not inward-locked) 20DraNramp. We propose a model in which metal and proton enter the transporter via the same 21 external pathway to the binding site, but follow separate routes to the cytoplasm, thus resolving 22 the electrostatic dilemma of using a cation co-substrate to drive a cation primary substrate. Our 23 results illustrate the flexibility of the LeuT fold to support a broad range of co-substrate coupling 24 and conformational change mechanisms. 25 Krishnamurthy and Gouaux, 2012;Malinauskaite et al., 2014;Ressl et al., 2009; Shimamura et 48 al., 2010; Weyand et al., 2008). 49Natural resistance-associated macrophage proteins (Nramps) are APC-superfamily transition 50 metal transporters that enable uptake of rare micronutrients such as Mn 2+ in plants and bacteria 51 and Fe 2+ in animals (Cellier, 2012;Courville et al., 2006;Nevo and Nelson, 2006). Nramps bind 52 and/or transport biologically essential divalent metals such as Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ -53 and toxic metals like Cd 2+ , Pb 2+ , and Hg 2+ -but not the abundant alkaline earth metals Mg 2+ and 54 Ca 2+ (Bozzi et al., 2016a;Ehrnstorfer et al., 2014). Metal uptake by Nramps involves proton co-55 transport, and many homologs also display considerable proton uniport-proton transport in the 56 absence of added metal that suggests loose coupling between the co-substrates (Chen et al., 1999; 57 Gunshin et al., 1997;Mackenzie et al., 2006;Nelson et al., 2002;Xu et al., 2004). Nramps have 58 11 or 12 TMs, the first ten forming a LeuT fold, as seen in structures of three bacterial Nramp 59 homologs (Bozzi et al., 2016b;Ehrnstorfer et al., 2014;Ehrnstorfer et al., 2017), including our 60 model system Deinococcus radiodurans (Dra)Nramp (Bozzi et al., 2016b). Conserved aspartate, 61 asparagine, and methionine residues in TM1 and TM6 coordinate transition metal substrates as 62 observed in an inward-open state (Ehrnstorfer et al., 2014), while only a metal-free outward-open 63 state has been reported (Ehrnstorfer et al., 2017). 64 Here we provide the first complementary structures of the same Nramp homolog in multiple 65 conformations, including the first metal-bound outward-open Nramp structure, and a novel 66inward-occluded structure. These allow us to fully illustrate the transport cycle for DraNramp. We 67 also show that metal transport requires the expected alternating access bulk confor...