Kanagalaghatta R. Rajashankar

Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP‐binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide‐binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF‐causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP‐ and ATP‐bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1‐transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1–NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

Molecular mechanism of membrane constriction and tubulation mediated by the F-BAR protein Pacsin/Syndapin

Peripheral membrane proteins of the Bin/amphiphysin/Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) family participate in cellular membrane trafficking and have been shown to generate membrane tubules. The degree of membrane bending appears to be encoded in the structure and immanent curvature of the particular protein domains, with BAR and F-BAR domains inducing high- and low-curvature tubules, respectively. In addition, oligomerization and the formation of ordered arrays influences tubule stabilization. Here, the F-BAR domain-containing protein Pacsin was found to possess a unique activity, creating small tubules and tubule constrictions, in addition to the wide tubules characteristic for this subfamily. Based on crystal structures of the F-BAR domain of Pacsin and mutagenesis studies, vesiculation could be linked to the presence of unique structural features distinguishing it from other F-BAR proteins. Tubulation was suppressed in the context of the full-length protein, suggesting that Pacsin is autoinhibited in solution. The regulated deformation of membranes and promotion of tubule constrictions by Pacsin suggests a more versatile function of these proteins in vesiculation and endocytosis beyond their role as scaffold proteins.

Host cell recognition by the henipaviruses: crystal structures of the Nipah G attachment glycoprotein and its complex with ephrin-B3.

Nipah virus (NiV) and Hendra virus are the type species of the highly pathogenic paramyxovirus genus Henipavirus, which can cause severe respiratory disease and fatal encephalitis infections in humans, with case fatality rates approaching 75%. NiV contains two envelope glycoproteins, the receptor-binding G glycoprotein (NiV-G) that facilitates attachment to host cells and the fusion (F) glycoprotein that mediates membrane merger. The henipavirus G glycoproteins lack both hemagglutinating and neuraminidase activities and, instead, engage the highly conserved ephrin-B2 and ephrin-B3 cell surface proteins as their entry receptors. Here, we report the crystal structures of the NiV-G both in its receptor-unbound state and in complex with ephrin-B3, providing, to our knowledge, the first view of a paramyxovirus attachment complex in which a cellular protein is used as the virus receptor. Complex formation generates an extensive protein–protein interface around a protruding ephrin loop, which is inserted in the central cavity of the NiV-G β-propeller. Analysis of the structural data reveals the molecular basis for the highly specific interactions of the henipavirus G glycoproteins with only two members (ephrin-B2 and ephrin-B3) of the very large ephrin family and suggests how they mediate in a unique fashion both cell attachment and the initiation of membrane fusion during the virus infection processes. The structures further suggest that the NiV-G/ephrin interactions can be effectively targeted to disrupt viral entry and provide the foundation for structure-based antiviral drug design.

Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same β-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein–protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the β-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

Crystal structure of a potassium ion transporter, TrkH

The TrkH/TrkG/KtrB proteins mediate K+ uptake in bacteria and probably evolved from simple K+ channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of K+ channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of K+ and Rb+ over smaller ions such as Na+ or Li+. Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of K+ flux. These results reveal the molecular basis of K+ selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins.

Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation.

CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids, using an RNA-mediated interference mechanism. Interference in type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine, Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Thermobifida fusca Cas3 bound to single-stranded (ss) DNA substrate and show that it is an obligate 3′-to-5′ ssDNase that preferentially accepts substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ssDNA cleavage. We demonstrate ATP coordination and conformational flexibility of the SF2-type helicase domain. Cas3 is specifically guided toward Cascade-bound target DNA by a PAM sequence, through physical interactions with both the nontarget substrate strand and the CasA protein. The sequence of recognition events ensures well-controlled DNA targeting and degradation of foreign DNA by Cascade and Cas3.

Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism

Dissecting how signaling directs axon growth During development of the nervous system, nerve cells send out projections called axons that must be guided to their proper targets. Netrins are secreted proteins that bind to receptors to either attract or repel the growing axons. Xu et al. present x-ray structures that show that complexes of netrin with two different receptors, neogenin and DCC, have different architectures. How netrin signals remains to be understood in detail, but netrins ability to create different assemblies probably plays a role in the diverse signaling outcomes it mediates. Science, this issue p. 1275 The axon guidance protein netrin binds to two receptors with different architectures, providing a basis for diverse signaling outcomes. Netrins are secreted proteins that regulate axon guidance and neuronal migration. Deleted in colorectal cancer (DCC) is a well-established netrin-1 receptor mediating attractive responses. We provide evidence that its close relative neogenin is also a functional netrin-1 receptor that acts with DCC to mediate guidance in vivo. We determined the structures of a functional netrin-1 region, alone and in complexes with neogenin or DCC. Netrin-1 has a rigid elongated structure containing two receptor-binding sites at opposite ends through which it brings together receptor molecules. The ligand/receptor complexes reveal two distinct architectures: a 2:2 heterotetramer and a continuous ligand/receptor assembly. The differences result from different lengths of the linker connecting receptor domains fibronectin type III domain 4 (FN4) and FN5, which differs among DCC and neogenin splice variants, providing a basis for diverse signaling outcomes.

Crystal structure of the receptor-binding domain from newly emerged Middle East respiratory syndrome coronavirus

ABSTRACT The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 77 people, with a fatality rate of more than 50%. Alarmingly, the virus demonstrates the capability of human-to-human transmission, raising the possibility of global spread and endangering world health and economy. Here we have identified the receptor-binding domain (RBD) from the MERS-CoV spike protein and determined its crystal structure. This study also presents a structural comparison of MERS-CoV RBD with other coronavirus RBDs, successfully positioning MERS-CoV on the landscape of coronavirus evolution and providing insights into receptor binding by MERS-CoV. Furthermore, we found that MERS-CoV RBD functions as an effective entry inhibitor of MERS-CoV. The identified MERS-CoV RBD may also serve as a potential candidate for MERS-CoV subunit vaccines. Overall, this study enhances our understanding of the evolution of coronavirus RBDs, provides insights into receptor recognition by MERS-CoV, and may help control the transmission of MERS-CoV in humans.

Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of α- and PPII-helices

Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 Å) crystal structure of the A3VP1 fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 Å) through the interaction of two noncontiguous regions in the primary sequence. The A3 repeat of the alanine-rich domain adopts an extended α-helix that intertwines with the P1 repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A3 and P1 helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

Fluoride ion encapsulation by Mg2+ ions and phosphates in a fluoride riboswitch.

Significant advances in our understanding of RNA architecture, folding and recognition have emerged from structure–function studies on riboswitches, non-coding RNAs whose sensing domains bind small ligands and whose adjacent expression platforms contain RNA elements involved in the control of gene regulation. We now report on the ligand-bound structure of the Thermotoga petrophila fluoride riboswitch, which adopts a higher-order RNA architecture stabilized by pseudoknot and long-range reversed Watson–Crick and Hoogsteen A•U pair formation. The bound fluoride ion is encapsulated within the junctional architecture, anchored in place through direct coordination to three Mg2+ ions, which in turn are octahedrally coordinated to water molecules and five inwardly pointing backbone phosphates. Our structure of the fluoride riboswitch in the bound state shows how RNA can form a binding pocket selective for fluoride, while discriminating against larger halide ions. The T. petrophila fluoride riboswitch probably functions in gene regulation through a transcription termination mechanism.