rGAT, Nayak et al.

MD simulation trajectories of rGAT in lipid bilayer system with GABA molecule bound

Exploring the limits of sequence and structure in a variant βγ-crystallin domain of the protein absent in melanoma-1 (AIM1)

βγ-Crystallins belong to a superfamily of proteins in prokaryotes and eukaryotes that are based on duplications of a characteristic, highly conserved Greek key motif. Most members of the superfamily in vertebrates are structural proteins of the eye lens that contain four motifs arranged as two structural domains. Absent in melanoma 1 (AIM1), an unusual member of the superfamily whose expression is associated with suppression of malignancy in melanoma, contains 12 βγ-crystallin motifs in six domains. Some of these motifs diverge considerably from the canonical motif sequence. AIM1g1, the first βγ-crystallin domain of AIM1, is the most variant of βγ-crystallin domains currently known. In order to understand the limits of sequence variation on the structure, we report the crystal structure of AIM1g1 at 1.9 Å resolution. Despite having changes in key residues, the domain retains the overall βγ-crystallin fold. The domain also contains an unusual extended surface loop that significantly alters the shape of the domain and its charge profile. This structure illustrates the resilience of the βγ fold to considerable sequence changes and its remarkable ability to adapt for novel functions

Transporters Through the Looking Glass: An Insight into the Mechanisms of Ion-Coupled Transport and Methods That Help Reveal Them

Cell membranes, despite providing a barrier to protect intracellular constituents, require selective gating for the influx of important metabolites including ions, sugars, amino acids, neurotransmitters and efflux of toxins and metabolic end-products. The machinery involved in carrying out this gating process comprises of integral membrane proteins that use ionic electrochemical gradients or ATP hydrolysis, to drive concentrative uptake or efflux. The mechanism through which ion-coupled transporters function is referred to as alternating-access. In the recent past, discrete modes of alternating-access have been described with the elucidation of new transporter structures and their snapshots in altered conformational states. Despite X-ray structures being the primary sources of mechanistic information, other biophysical methods provide information related to the structural dynamics of these transporters. Methods including EPR and smFRET, have extensively helped validate or clarify ion-coupled transport mechanisms, in a near-native environment. This review seeks to highlight the mechanistic details of ion-coupled transport and delve into the biophysical tools and methods that help in understanding these fascinating molecules

Source_file.xlsxCryo-EM structures of Pannexin 1 and 3 reveal differences among Pannexin isoforms

Pannexins are single-membrane large-pore ion channels that releases ions and ATP upon activation. Three isoforms of pannexins 1, 2, and 3, perform diverse cellular roles and differ in their pore lining residues. In this study, we report the cryo-EM structure of pannexin 3 at 3.9 Å and analyze its structural differences with pannexin isoforms 1 and 2. The pannexin 3 vestibule has two distinct chambers and a wider pore radius in comparison to pannexins 1 and 2. We further report two cryo-EM structures of pannexin 1, with pore substitutions W74R/R75D that mimic the pore lining residues of pannexin 2 and a germline mutant of pannexin 1, R217H at resolutions of 3.2 Å and 3.9 Å, respectively. Substitution of cationic residues in the vestibule of pannexin 1 results in reduced ATP interaction propensities to the channel. The germline mutant R217H in transmembrane helix 3 (TM3), leads to a partially constricted pore, reduced ATP interaction and weakened voltage sensitivity. The study compares the thre pannexin isoform structures, the effects of substitutions of pore and vestibule-lining residues and allosteric effects of a pathological substitution on channel structure and function thereby enhancing our understanding of this vital group of ATP-release channels.</p

Dissection of Protonation Sites for Antibacterial Recognition and Transport in QacA, a Multi-Drug Efflux Transporter

QacA is a drug:H+ antiporter with 14 transmembrane helices that confers antibacterial resistance to methicillin-resistant Staphylococcus aureus strains, with homologs in other pathogenic organisms. It is a highly promiscuous antiporter, capable of H+-driven efflux of a wide array of cationic antibacterial compounds and dyes. Our study, using a homology model of QacA, reveals a group of six protonatable residues in its vestibule. Systematic mutagenesis resulted in the identification of D34 (TM1), and a cluster of acidic residues in TM13 including E407 and D411 and D323 in TM10, as being crucial for substrate recognition and transport of monovalent and divalent cationic antibacterial compounds. The transport and binding properties of QacA and its mutants were explored using whole cells, inside-out vesicles, substrate-induced H+ release and microscale thermophoresis-based assays. The activity of purified QacA was also observed using proteoliposome-based substrate-induced H+ transport assay. Our results identify two sites, D34 and D411 as vital players in substrate recognition, while E407 facilitates substrate efflux as a protonation site. We also observe that E407 plays an additional role as a substrate recognition site for the transport of dequalinium, a divalent quaternary ammonium compound. These observations rationalize the promiscuity of QacA for diverse substrates. The study unravels the role of acidic residues in QacA with implications for substrate recognition, promiscuity and processive transport in multidrug efflux transporters, related to QacA

Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg<SUP>2+</SUP> modulates Ca<SUP>2+</SUP> binding, Ca<SUP>2+</SUP>- induced conformational changes, and equilibrium unfolding transitions

Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca<SUP>2+</SUP> signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca<SUP>2+</SUP>-specific) and structural (Ca<SUP>2+</SUP>- or Mg<SUP>2+</SUP>-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca<SUP>2+</SUP> binding using NMR and EF-hand mutants. Ca<SUP>2+</SUP> titration, as monitored by [<SUP>15</SUP>N,<SUP>1</SUP>H] heteronuclear single quantum coherence, suggests that Ca<SUP>2+</SUP> binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg<SUP>2+</SUP> binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca<SUP>2+</SUP>-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca<SUP>2+</SUP> and not Mg<SUP>2+</SUP>. Ca<SUP>2+</SUP> binding induces conformational rearrangements in the protein by reversing Mg<SUP>2+</SUP>-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg<SUP>2+</SUP> with Ca<SUP>2+</SUP> reduces the Ca<SUP>2+</SUP>-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca<SUP>2+</SUP>-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg<SUP>2+</SUP>-bound NCS-1 are similar, the early unfolding transitions of Ca<SUP>2+</SUP>-bound NCS-1 are partially influenced in the presence of Mg<SUP>2+</SUP>. This study demonstrates the importance of Mg<SUP>2+</SUP> as a modulator of calcium homeostasis and active-state behavior of NCS-1

The βγ-crystallin superfamily contains a Universal motif for binding calcium

The βγ-crystallin superfamily consists of evolutionarily related proteins with domain topology similar to lens β- and γ-crystallins, formed from duplicated Greek key motifs. Ca2+ binding was found in a few βγ-crystallin members earlier, although its prevalence and diversity as inherent molecular properties among members of the superfamily are not well studied. To increase our understanding of Ca2+ binding in various βγ-crystallins, we undertook comprehensive structural and Ca2+-binding studies of seven members of the superfamily from bacteria, archaea, and vertebrates, including determination of high-resolution crystal structures of three proteins. Our structural observations show that the determinants of Ca2+ coordination remain conserved in the form of an N/D-N/D-#-I-S/T-S motif in all domains. However, binding of Ca2+ elicits varied physicochemical responses, ranging from passive sequestration to active stabilization. The motif in this superfamily is modified in some members like lens crystallins where Ca2+-binding abilities are partly or completely compromised. We show that reduction or loss of Ca2+ binding in members of the superfamily, particularly in vertebrates, is due to the selective presence of unfavorable amino acids (largely Arg) at key Ca2+-ligation positions and that engineering of the canonical Ca2+-binding residues can confer binding activity on an otherwise inactive domain. Through this work, we demonstrate that βγ-crystallins with the N/D-N/D-#-I-S/T-S motif form an extensive set of Ca2+-binding proteins prevalent in all of the three kingdoms of life