Role of TlyA in the Biology of Uncultivable Mycobacteria

TlyA proteins are related to distinct functions in a diverse spectrum of bacterial pathogens including mycobacterial spp. There are several annotated proteins function as hemolysin or pore forming molecules that play an important role in the virulence of pathogenic organisms. Many studies reported the dual activity of mycobacterial TlyA as ‘hemolysin’ and ‘S-adenosylmethionine dependent rRNA methylase’. To act as a hemolysin, a sequence must have a signal sequence and transmembrane segment which helps the protein to enter the extracellular environment. Interestingly, the mycobacterial tlyA has neither a traditional signal sequences of general/sec/tat pathways nor any transmembrane segments are present. Still it can reach the extracellular milieu with the help of non-classical signal mechanisms. Also, retention of tlyA in cultivable mycobacterial pathogens (such as Mycobacterium tuberculosis and M. marinum) as well as uncultivated mycobacterial pathogens despite their extreme reductive evolution (such as M. leprae, M. lepromatosis and M. uberis) suggests its crucial role in evolutionary biology of pathogenic mycobacteria. Numerous virulence factors have been characterised from the uncultivable mycobacteria but the information of TlyA protein is still limited in terms of molecular and structural characterisation. The genomic insights offered by comparative analysis of TlyA sequences and its conserved domains reveal its pore forming activity which further confirms its role as a virulence protein, particularly in uncultivable mycobacteria. Therefore, this review presents a comparative analysis of mycobacterial TlyA family by sequence homology and alignment to improve our understanding of this unconventional hemolysin and RNA methyltransferase TlyA of uncultivable mycobacteria.

Conserved Proline Residues of Bacillus subtilis Intramembrane Metalloprotease SpoIVFB Are Important for Substrate Interaction and Cleavage

Intramembrane metalloproteases regulate diverse biological processes by cleaving membrane-associated substrates within the membrane or near its surface. SpoIVFB is an intramembrane metalloprotease of Bacillus subtilis that cleaves Pro-σ K during endosporulation. Intramembrane metalloproteases have a broadly conserved NPDG motif, which in the structure of an archaeal enzyme is located in a short loop that interrupts a transmembrane segment facing the active site. The aspartate residue of the NPDG motif acts as a ligand of the zinc ion involved in catalysis. The functions of other residues in the short loop are less well understood. We found that the predicted short loop of SpoIVFB contains two highly conserved proline residues, P132 of the NPDG motif and P135. Mutational analysis revealed that both proline residues are important for Pro-σ K cleavage in Escherichia coli engineered to synthesize the proteins. Substitutions for either residue also impaired Pro-σ K interaction with SpoIVFB in co-purification assays. Disulfide cross-linking experiments showed that the predicted short loop of SpoIVFB is in proximity to the Proregion of Pro-σ K . Alanine substitutions for N129 and P132 of the SpoIVFB NPDG motif reduced cross-linking between its predicted short loop and the Proregion more than a P135A substitution. Conversely, the SpoIVFB P135A substitution reduced Pro-σ K cleavage more than the N129A and P132A substitutions during sporulation of B. subtilis . We conclude that all three conserved residues of SpoIVFB are important for substrate interaction and cleavage, and we propose that P135 is necessary to position D137 to act as a zinc ligand. IMPORTANCE Intramembrane metalloproteases (IMMPs) function in numerous signaling pathways. Bacterial IMMPs govern stress responses, including sporulation of some species, thus enhancing the virulence and persistence of pathogens. Knowledge of IMMP-substrate interactions could aid therapeutic design, but structures of IMMP·substrate complexes are unknown. We examined interaction of the IMMP SpoIVFB with its substrate Pro-σ K , whose cleavage is required for Bacillus subtilis endosporulation. We found that conserved proline residues in a short loop predicted to interrupt a SpoIVFB transmembrane segment are important for Pro-σ K binding and cleavage. Corresponding residues of the Escherichia coli IMMP RseP have also been shown to be important for substrate interaction and cleavage, suggesting this is a broadly conserved feature of IMMPs, potentially suitable as a therapeutic target.

The Conformationally Sensitive Spatial Distance Between the TM3-4 Loop and Transmembrane Segment 7 in the Glutamate Transporter Revealed by Paired-Cysteine Mutagenesis

Excitatory amino acid transporters can maintain extracellular glutamate concentrations lower than neurotoxic levels by transferring neurotransmitters from the synaptic cleft into surrounding glial cells and neurons. Previous work regarding the structural studies of GltPh, GltTK, excitatory amino acid transporter 1 (EAAT1), EAAT3 and alanine serine cysteine transporter 2 described the transport mechanism of the glutamate transporter in depth. However, much remains unknown about the role of the loop between transmembrane segment 3 and 4 during transport. To probe the function of this loop in the transport cycle, we engineered a pair of cysteine residues between the TM3-TM4 loop and TM7 in cysteine-less EAAT2. Here, we show that the oxidative cross-linking reagent CuPh inhibits transport activity of the paired mutant L149C/M414C, whereas DTT inhibits the effect of CuPh on transport activity of L149C/M414C. Additionally, we show that the effect of cross-linking in the mutant is due to the formation of the disulfide bond within the molecules of EAAT2. Further, L-glutamate or KCl protect, and D,L-threo-β-benzyloxy-aspartate (TBOA) increases, CuPh-induced inhibition in the L149C/M414 mutant, suggesting that the L149C and M414C cysteines are closer or farther away in the outward- or inward-facing conformations, respectively. Together, our findings provide evidence that the distance between TM3-TM4 loop and TM7 alter when substrates are transported.

A second S4 movement opens hyperpolarization-activated HCN channels

Rhythmic activity in pacemaker cells, as in the sino-atrial node in the heart, depends on the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. As in depolarization-activated K+ channels, the fourth transmembrane segment S4 functions as the voltage sensor in hyperpolarization-activated HCN channels. But how the inward movement of S4 in HCN channels at hyperpolarized voltages couples to channel opening is not understood. Using voltage clamp fluorometry, we found here that S4 in HCN channels moves in two steps in response to hyperpolarizations and that the second S4 step correlates with gate opening. We found a mutation in sea urchin HCN channels that separate the two S4 steps in voltage dependence. The E356A mutation in S4 shifts the main S4 movement to positive voltages, but channel opening remains at negative voltages. In addition, E356A reveals a second S4 movement at negative voltages that correlates with gate opening. Cysteine accessibility and molecular models suggest that the second S4 movement opens up an intracellular crevice between S4 and S5 that would allow radial movement of the intracellular ends of S5 and S6 to open HCN channels.

Voltage-gated sodium channels (NaV) in GtoPdb v.2021.3

Sodium channels are voltage-gated sodium-selective ion channels present in the membrane of most excitable cells. Sodium channels comprise of one pore-forming α subunit, which may be associated with either one or two β subunits [177]. α-Subunits consist of four homologous domains (I-IV), each containing six transmembrane segments (S1-S6) and a pore-forming loop. The positively charged fourth transmembrane segment (S4) acts as a voltage sensor and is involved in channel gating. The crystal structure of the bacterial NavAb channel has revealed a number of novel structural features compared to earlier potassium channel structures including a short selectivity filter with ion selectivity determined by interactions with glutamate side chains [274]. Interestingly, the pore region is penetrated by fatty acyl chains that extend into the central cavity which may allow the entry of small, hydrophobic pore-blocking drugs [274]. Auxiliary β1, β2, β3 and β4 subunits consist of a large extracellular N-terminal domain, a single transmembrane segment and a shorter cytoplasmic domain.The nomenclature for sodium channels was proposed by Goldin et al., (2000) [144] and approved by the NC-IUPHAR Subcommittee on sodium channels (Catterall et al., 2005, [52]).

The M1 and pre-M1 segments contribute differently to ion selectivity in ASICs and ENaCs

The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium ratio ∼10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). This discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs, and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we used conventional and noncanonical amino acid–based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that these regions contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work suggests that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. These results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.

Plasma Membrane Ca2+ Pump PMCA4z Is More Active Than Splicing Variant PMCA4x

The plasma membrane Ca2+ pumps (PMCA) are P-ATPases that control Ca2+ signaling and homeostasis by transporting Ca2+ out of the eukaryotic cell. Humans have four genes that code for PMCA isoforms (PMCA1-4). A large diversity of PMCA isoforms is generated by alternative mRNA splicing at sites A and C. The different PMCA isoforms are expressed in a cell-type and developmental-specific manner and exhibit differential sensitivity to a great number of regulatory mechanisms. PMCA4 has two A splice variants, the forms “x” and “z”. While PMCA4x is ubiquitously expressed and relatively well-studied, PMCA4z is less characterized and its expression is restricted to some tissues such as the brain and heart muscle. PMCA4z lacks a stretch of 12 amino acids in the so-called A-M3 linker, a conformation-sensitive region of the molecule connecting the actuator domain (A) with the third transmembrane segment (M3). We expressed in yeast PMCA4 variants “x” and “z”, maintaining constant the most frequent splice variant “b” at the C-terminal end, and obtained purified preparations of both proteins. In the basal autoinhibited state, PMCA4zb showed a higher ATPase activity and a higher apparent Ca2+ affinity than PMCA4xb. Both isoforms were stimulated by calmodulin but PMCA4zb was more strongly activated by acidic lipids than PMCA4xb. The results indicate that a PMCA4 intrinsically more active and more responsive to acidic lipids is produced by the variant “z” of the splicing site A.

Efficient integration of transmembrane domains depends on the folding properties of the upstream sequences

The topology of most membrane proteins is defined by the successive integration of α-helical transmembrane domains at the Sec61 translocon. The translocon provides a pore for the transfer of polypeptide segments across the membrane while giving them lateral access to the lipid. For each polypeptide segment of ∼20 residues, the combined hydrophobicities of its constituent amino acids were previously shown to define the extent of membrane integration. Here, we discovered that different sequences preceding a potential transmembrane domain substantially affect its hydrophobicity requirement for integration. Rapidly folding domains, sequences that are intrinsically disordered or very short or capable of binding chaperones with high affinity, allow for efficient transmembrane integration with low-hydrophobicity thresholds for both orientations in the membrane. In contrast, long protein fragments, folding-deficient mutant domains, and artificial sequences not binding chaperones interfered with membrane integration, requiring higher hydrophobicity. We propose that the latter sequences, as they compact on their hydrophobic residues, partially folded but unable to reach a native state, expose hydrophobic surfaces that compete with the translocon for the emerging transmembrane segment, reducing integration efficiency. The results suggest that rapid folding or strong chaperone binding is required for efficient transmembrane integration.

Capture and delivery of tail-anchored proteins to the endoplasmic reticulum

Tail-anchored (TA) proteins fulfill diverse cellular functions within different organellar membranes. Their characteristic C-terminal transmembrane segment renders TA proteins inherently prone to aggregation and necessitates their posttranslational targeting. The guided entry of TA proteins (GET in yeast)/transmembrane recognition complex (TRC in humans) pathway represents a major route for TA proteins to the endoplasmic reticulum (ER). Here, we review important new insights into the capture of nascent TA proteins at the ribosome by the GET pathway pretargeting complex and the mechanism of their delivery into the ER membrane by the GET receptor insertase. Interestingly, several alternative routes by which TA proteins can be targeted to the ER have emerged, raising intriguing questions about how selectivity is achieved during TA protein capture. Furthermore, mistargeting of TA proteins is a fundamental cellular problem, and we discuss the recently discovered quality control machineries in the ER and outer mitochondrial membrane for displacing mislocalized TA proteins.

Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors

Serotonin receptors play important roles in neuronal excitation, emotion, platelet aggregation, and vasoconstriction. The serotonin receptor subtype 2A (5-HT2AR) is a Gq-coupled GPCR, which activate phospholipase C. Although the structures and functions of 5-HT2ARs have been well studied, little has been known about their real-time dynamics. In this study, we analyzed the intramolecular motion of the 5-HT2AR in living cells using the diffracted X-ray tracking (DXT) technique. The DXT is a very precise single-molecular analytical technique, which tracks diffraction spots from the gold nanocrystals labeled on the protein surface. Trajectory analysis provides insight into protein dynamics. The 5-HT2ARs were transiently expressed in HEK 293 cells, and the gold nanocrystals were attached to the N-terminal introduced FLAG-tag via anti-FLAG antibodies. The motions were recorded with a frame rate of 100 μs per frame. A lifetime filtering technique demonstrated that the unliganded receptors contain high mobility population with clockwise twisting. This rotation was, however, abolished by either a full agonist α-methylserotonin or an inverse agonist ketanserin. Mutation analysis revealed that the “ionic lock” between the DRY motif in the third transmembrane segment and a negatively charged residue of the sixth transmembrane segment is essential for the torsional motion at the N-terminus of the receptor.