Heterobimetallic Base Pair Programming in Designer 3D DNA Crystals.

Metal-mediated DNA (mmDNA) presents a pathway toward engineering bioinorganic and electronic behavior into DNA devices. Many chemical and biophysical forces drive the programmable chelation of metals between pyrimidine base pairs. Here, we developed a crystallographic method using the three-dimensional (3D) DNA tensegrity triangle motif to capture single- and multi-metal binding modes across granular changes to environmental pH using anomalous scattering. Leveraging this programmable crystal, we determined 28 biomolecular structures to capture mmDNA reactions. We found that silver(I) binds with increasing occupancy in T-T and U-U pairs at elevated pH levels, and we exploited this to capture silver(I) and mercury(II) within the same base pair and to isolate the titration points for homo- and heterometal base pair modes. We additionally determined the structure of a C-C pair with both silver(I) and mercury(II). Finally, we extend our paradigm to capture cadmium(II) in T-T pairs together with mercury(II) at high pH. The precision self-assembly of heterobimetallic DNA chemistry at the sub-nanometer scale will enable atomistic design frameworks for more elaborate mmDNA-based nanodevices and nanotechnologies.

Metal-Mediated DNA Nanotechnology in 3D: Structural Library by Templated Diffraction.

DNA double helices containing metal-mediated DNA (mmDNA) base pairs are constructed from Ag+ and Hg2+ ions between pyrimidine:pyrimidine pairs with the promise of nanoelectronics. Rational design of mmDNA nanomaterials is impractical without a complete lexical and structural description. Here, the programmability of structural DNA nanotechnology toward its founding mission of self-assembling a diffraction platform for biomolecular structure determination is explored. The tensegrity triangle is employed to build a comprehensive structural library of mmDNA pairs via X-ray diffraction and generalized design rules for mmDNA construction are elucidated. Two binding modes are uncovered: N3-dominant, centrosymmetric pairs and major groove binders driven by 5-position ring modifications. Energy gap calculations show additional levels in the lowest unoccupied molecular orbitals (LUMO) of mmDNA structures, rendering them attractive molecular electronic candidates.

Metal-Mediated DNA Nanotechnology in 3D: Structural Library by Templated Diffraction.

DNA double helices containing metal-mediated DNA (mmDNA) base pairs have been constructed from Ag+ and Hg2+ ions between pyrimidine:pyrimidine pairs with the promise of nanoelectronics. Rational design of mmDNA nanomaterials has been impractical without a complete lexical and structural description. Here, we explore the programmability of structural DNA nanotechnology toward its founding mission of self-assembling a diffraction platform for biomolecular structure determination. We employed the tensegrity triangle to build a comprehensive structural library of mmDNA pairs via X-ray diffraction and elucidated generalized design rules for mmDNA construction. We uncovered two binding modes: N3-dominant, centrosymmetric pairs and major groove binders driven by 5-position ring modifications. Energy gap calculations showed additional levels in the lowest unoccupied molecular orbitals (LUMO) of mmDNA structures, rendering them attractive molecular electronic candidates. This article is protected by copyright. All rights reserved.

Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms.

Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired crossreactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ∼ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.

NaV1.2 EFL domain allosterically enhances Ca2+ binding to sites I and II of WT and pathogenic calmodulin mutants bound to the channel CTD.

Neuronal voltage-gated sodium channel NaV1.2 C-terminal domain (CTD) binds calmodulin (CaM) constitutively at its IQ motif. A solution structure (6BUT) and other NMR evidence showed that the CaM N domain (CaMN) is structurally independent of the C-domain (CaMC) whether CaM is bound to the NaV1.2IQp (1,901-1,927) or NaV1.2CTD (1,777-1,937) with or without calcium. However, in the CaM + NaV1.2CTD complex, the Ca2+ affinity of CaMN was more favorable than in free CaM, while Ca2+ affinity for CaMC was weaker than in the CaM + NaV1.2IQp complex. The CTD EF-like (EFL) domain allosterically widened the energetic gap between CaM domains. Cardiomyopathy-associated CaM mutants (N53I(N54I), D95V(D96V), A102V(A103V), E104A(E105A), D129G(D130G), and F141L(F142L)) all bound the NaV1.2 IQ motif favorably under resting (apo) conditions and bound calcium normally at CaMN sites. However, only N53I and A102V bound calcium at CaMC sites at [Ca2+] < 100 µM. Thus, they are expected to respond like wild-type CaM to Ca2+ spikes in excitable cells.

Structural basis of cytoplasmic NaV1.5 and NaV1.4 regulation.

Voltage-gated sodium channels (NaVs) are membrane proteins responsible for the rapid upstroke of the action potential in excitable cells. There are nine human voltage-sensitive NaV1 isoforms that, in addition to their sequence differences, differ in tissue distribution and specific function. This review focuses on isoforms NaV1.4 and NaV1.5, which are primarily expressed in skeletal and cardiac muscle cells, respectively. The determination of the structures of several eukaryotic NaVs by single-particle cryo-electron microscopy (cryo-EM) has brought new perspective to the study of the channels. Alignment of the cryo-EM structure of the transmembrane channel pore with x-ray crystallographic structures of the cytoplasmic domains illustrates the complementary nature of the techniques and highlights the intricate cellular mechanisms that modulate these channels. Here, we review structural insights into the cytoplasmic C-terminal regulation of NaV1.4 and NaV1.5 with special attention to Ca2+ sensing by calmodulin, implications for disease, and putative channel dimerization.

Ca2+-dependent regulation of sodium channels NaV1.4 and NaV1.5 is controlled by the post-IQ motif.

Skeletal muscle voltage-gated Na+ channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2+-dependent inactivation; no such inactivation is observed in the cardiac Na+ channel (NaV1.5). Taken together, the crystal structures of the NaV1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca2+-dependent CaM N-lobe binding site previously identified in NaV1.5 is not present in NaV1.4 allowing the N-lobe to signal other regions of the NaV1.4 channel. Consistent with this mechanism, removing this binding site in NaV1.5 unveils robust Ca2+-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca2+-dependent inactivation is effected by CaM's N-lobe binding outside the NaV C-terminal while CaM's C-lobe remains bound to the NaV C-terminal. As the N-lobe binding motif of NaV1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.

Bilobal architecture is a requirement for calmodulin signaling to CaV1.3 channels.

Calmodulin (CaM) regulation of voltage-gated calcium (CaV) channels is a powerful Ca2+ feedback mechanism that adjusts Ca2+ influx, affording rich mechanistic insights into Ca2+ decoding. CaM possesses a dual-lobed architecture, a salient feature of the myriad Ca2+-sensing proteins, where two homologous lobes that recognize similar targets hint at redundant signaling mechanisms. Here, by tethering CaM lobes, we demonstrate that bilobal architecture is obligatory for signaling to CaV channels. With one lobe bound, CaV carboxy tail rearranges itself, resulting in a preinhibited configuration precluded from Ca2+ feedback. Reconstitution of two lobes, even as separate molecules, relieves preinhibition and restores Ca2+ feedback. CaV channels thus detect the coincident binding of two Ca2+-free lobes to promote channel opening, a molecular implementation of a logical NOR operation that processes spatiotemporal Ca2+ signals bifurcated by CaM lobes. Overall, a unified scheme of CaV channel regulation by CaM now emerges, and our findings highlight the versatility of CaM to perform exquisite Ca2+ computations.

Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel NaV1.2.

Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel NaV1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat NaV1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3nM) and (Ca2+)4-CaM (~85nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca2+)4-CaM, NMR demonstrated that NaV1.2 IQ motif peptide (NaV1.2IQp) exclusively made contacts with C-domain residues of CaM (CaMC). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca2+)2-CaMC bound to NaV1.2IQp. The polarity of (Ca2+)2-CaMC relative to the IQ motif was opposite to that seen in apo CaMC-Nav1.2IQp (2KXW), revealing that CaMC recognizes nested, anti-parallel sites in Nav1.2IQp. Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaMN allowing interactions with non-IQ NaV1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif.

Calmodulin and Ca(2+) control of voltage gated Na(+) channels.

The structures of the cytosolic portion of voltage activated sodium channels (CTNav) in complexes with calmodulin and other effectors in the presence and the absence of calcium provide information about the mechanisms by which these effectors regulate channel activity. The most studied of these complexes, those of Nav1.2 and Nav1.5, show details of the conformations and the specific contacts that are involved in channel regulation. Another voltage activated sodium channel, Nav1.4, shows significant calcium dependent inactivation, while its homolog Nav1.5 does not. The available structures shed light on the possible localization of the elements responsible for this effect. Mutations in the genes of these 3 Nav channels are associated with several disease conditions: Nav1.2, neurological conditions; Nav1.4, syndromes involving skeletal muscle; and Nav1.5, cardiac arrhythmias. Many of these disease-specific mutations are located at the interfaces involving CTNav and its effectors.