A common regulatory mechanism for methyltransferases involves the formation of complexes with their closely related counterparts. Previously, we found that METTL11A (NRMT1/NTMT1), an N-trimethylase, is activated by binding to its close homolog METTL11B (NRMT2/NTMT2). In further reports, METTL11A is observed co-fractionating with METTL13, a third METTL family member, modifying both the N-terminus and lysine 55 (K55) of the eukaryotic elongation factor 1 alpha protein. We confirm a regulatory interaction between METTL11A and METTL13 using co-immunoprecipitation, mass spectrometry, and in vitro methylation assays. Our findings show METTL11B enhances METTL11A's activity, while METTL13 inhibits it. This marks the first instance where a methyltransferase is observed to be controlled in an opposing fashion by various members of the same family. A similar outcome is noted, where METTL11A stimulates METTL13's K55 methylation activity, but at the same time, it hinders its N-methylation capacity. These regulatory impacts, as we have determined, do not necessitate catalytic activity, revealing new, non-catalytic roles for METTL11A and METTL13. The final demonstration shows that METTL11A, METTL11B, and METTL13 can collectively form a complex, and in the presence of all three, the regulatory influence of METTL13 outweighs that of METTL11B. These observations afford a deeper insight into the regulation of N-methylation, prompting a model wherein these methyltransferases may function in both catalytic and noncatalytic capacities.
The synaptic development process is influenced by MDGAs (MAM domain-containing glycosylphosphatidylinositol anchors), synaptic cell-surface molecules that are instrumental in establishing trans-synaptic bridges between neurexins (NRXNs) and neuroligins (NLGNs). Various neuropsychiatric illnesses are associated with alterations in MDGA genes. On the postsynaptic membrane, MDGAs form cis-binding interactions with NLGNs, obstructing their subsequent binding to NRXNs. MDGA1's crystal structure, showcasing six immunoglobulin (Ig) and one fibronectin III domain, reveals a striking, compact, triangular arrangement, both in its free state and when bound to NLGNs. The question of whether this unusual domain arrangement is crucial for biological function, or if alternative arrangements exhibit distinct functional outcomes, remains unresolved. The present research indicates that WT MDGA1 is capable of binding NLGN2 through its dynamic three-dimensional structure, which can adopt either a compact or an extended conformation. Strategic molecular elbows in MDGA1 are manipulated by designer mutants, leading to changes in the distribution of 3D conformations, while keeping the binding affinity of MDGA1's soluble ectodomains and NLGN2 constant. Within a cellular framework, these mutants present unusual combinations of functional outcomes, including altered binding to NLGN2, reduced capacity for concealing NLGN2 from NRXN1, and/or dampened NLGN2-mediated inhibitory presynaptic maturation, despite the mutations' location apart from the MDGA1-NLGN2 interaction site. Gamcemetinib Consequently, the 3D structure of the complete MDGA1 ectodomain appears crucial for its function, and the NLGN binding site within Ig1-Ig2 is not isolated from the complete molecule. A molecular mechanism to regulate MDGA1 function in the synaptic cleft may be based on 3D conformational changes within the MDGA1 ectodomain, particularly through the influence of strategic elbow points.
Myosin regulatory light chain 2 (MLC-2v) phosphorylation directly affects the degree to which cardiac contraction is controlled. The equilibrium between the activities of MLC kinases and phosphatases establishes the phosphorylation level of MLC-2v. Myosin Phosphatase Targeting Subunit 2 (MYPT2) is a key component of the MLC phosphatase predominantly observed in cardiac muscle cells. Cardiac myocyte MYPT2 overexpression leads to a decrease in MLC phosphorylation, a reduction in left ventricular contraction strength, and hypertrophy development; the effect of MYPT2 deletion on cardiac performance, however, is yet to be elucidated. From the Mutant Mouse Resource Center, we obtained heterozygous mice harboring a null allele of MYPT2. Cardiac myocytes in these mice, originating from a C57BL/6N background, were deficient in MLCK3, the principal regulatory light chain kinase. We observed that MYPT2-deficient mice exhibited complete viability and no observable phenotypic variations when compared to the wild-type control group. Our investigation also determined that WT C57BL/6N mice displayed a reduced basal level of MLC-2v phosphorylation, which was substantially enhanced when MYPT2 was not present. At the 12-week mark, the hearts of MYPT2-knockout mice were smaller, revealing diminished expression of genes pertinent to cardiac structural modification. Cardiac ultrasound analysis of 24-week-old male MYPT2 knockout mice indicated a diminished heart size and an improved fractional shortening, relative to their MYPT2 wild-type littermates. A synthesis of these studies reveals MYPT2's critical role in cardiac function in vivo, and its deletion is shown to partially compensate for the deficiency of MLCK3.
Mycobacterium tuberculosis (Mtb)'s sophisticated type VII secretion system is instrumental in transporting virulence factors across its intricate lipid membrane. Secreted by the ESX-1 apparatus, EspB, a protein of 36 kDa, was shown to instigate host cell death, an effect separate from ESAT-6. Although a substantial amount of high-resolution structural data exists for the ordered N-terminal domain, the precise mechanism of EspB-mediated virulence is not yet fully understood. Through a biophysical lens, incorporating transmission electron microscopy and cryo-electron microscopy, we present the details of EspB's engagement with phosphatidic acid (PA) and phosphatidylserine (PS) within the context of membranes. The conversion of monomers to oligomers, governed by PA and PS, was observed at a physiological pH. Gamcemetinib Our analysis indicates that EspB displays a restricted association with biological membranes, primarily interacting with phosphatidic acid (PA) and phosphatidylserine (PS). EspB's effect on yeast mitochondria implies a mitochondrial membrane-binding aptitude for this ESX-1 substrate. Furthermore, the three-dimensional structures of EspB, in the presence and absence of PA, were determined, revealing a likely stabilization of the low-complexity C-terminal domain when PA was involved. Our cryo-EM structural and functional studies of EspB, taken together, deepen our understanding of how Mycobacterium tuberculosis interacts with its host.
The bacterium Serratia proteamaculans is the source of Emfourin (M4in), a newly identified protein metalloprotease inhibitor that serves as the prototype for a novel class of protein protease inhibitors, the exact mechanism of which is yet to be determined. Protealysin-like proteases (PLPs) of the thermolysin family are natural substrates for emfourin-like inhibitors, commonly found in bacterial and archaeal species. Analysis of the available data suggests a role for PLPs in bacterial-bacterial interactions, interactions between bacteria and other life forms, and possibly in the development of disease. Control of PLP activity is potentially mediated by emfourin-like inhibitors, thereby influencing the course of bacterial diseases. Solution NMR spectroscopic methods were utilized to ascertain the 3D structure of the M4in protein. The observed structure displayed no substantial similarity to any cataloged protein structures. This structure was instrumental in constructing a model of the M4in-enzyme complex, which was confirmed through the use of small-angle X-ray scattering. Based on the model analysis, we present a molecular mechanism underlying the inhibitor's action, which has been validated by site-directed mutagenesis. Our findings underscore the pivotal role of two proximate, flexible loop domains in facilitating the interaction between the inhibitor and the protease. A coordination bond with the enzyme's catalytic Zn2+ is formed by aspartic acid in one region, contrasting with the second region housing hydrophobic amino acids that engage with the protease's substrate binding sites. The active site's specific structure is associated with a non-canonical inhibition process. The initial demonstration of such a mechanism for thermolysin family metalloprotease protein inhibitors highlights M4in as a novel foundation for antibacterial agent development, targeting selective inhibition of key bacterial pathogenesis factors within this family.
In the context of multiple critical biological pathways, including transcriptional activation, DNA demethylation, and DNA repair, thymine DNA glycosylase (TDG) acts as a multifaceted enzyme. Recent findings have exposed regulatory ties between TDG and RNA, however, the exact molecular interactions at the heart of these connections are not yet fully understood. Direct binding of TDG to RNA, with nanomolar affinity, is now demonstrated. Gamcemetinib Employing synthetic oligonucleotides of specific length and sequence, we establish TDG's strong predilection for G-rich sequences in single-stranded RNA, demonstrating minimal binding to single-stranded DNA and duplex RNA. Endogenous RNA sequences are also tightly bound by TDG. Truncated protein studies reveal that the structured catalytic domain of TDG is the primary RNA-binding site, while the disordered C-terminal domain significantly influences TDG's RNA affinity and selectivity. The competition between RNA and DNA for TDG binding is presented, ultimately showing that RNA presence impairs TDG's ability to catalyze excision. The findings of this study lend support to and offer insights into a mechanism wherein TDG-mediated procedures (such as DNA demethylation) are regulated by the direct engagement of TDG with RNA.
Foreign antigens are presented to T cells by dendritic cells (DCs) through the major histocompatibility complex (MHC), thereby initiating acquired immune responses. Local inflammatory responses are frequently initiated by the accumulation of ATP in inflamed areas or in tumor tissues. Nonetheless, the precise mechanism by which ATP influences dendritic cell function warrants further investigation.