Terminal Loop Research Articles

Structure of Essential RNA Regulatory Elements in the West Nile Virus 3′-Terminal Stem Loop

Flaviviruses, such as West Nile and Dengue Virus, pose a significant and growing threat to global health. Central to the flavivirus life cycle are highly structured 5′- and 3′-untranslated regions (UTRs), which harbor conserved cis-acting RNA elements critical for viral replication and host adaptation. Despite their essential roles, detailed molecular insights into these RNA elements have been limited. By employing nuclear magnetic resonance (NMR) spectroscopy in conjunction with SAXS experiments, we determined the three-dimensional structure of the West Nile Virus (WNV) 3′-terminal stem-loop core, a highly conserved element critical for viral genome cyclization and replication. Single nucleotide mutations at several sites within this RNA abolish the ability of the virus to replicate. These critical sites are located within a short 18-nucleotide hairpin stem, a substructure notable for its conformational flexibility, while the adjoining main stem-loop adopts a well-defined extended helix interrupted by three non-Watson-Crick pairs. This study enhances our understanding of several metastable RNA structures that play key roles in regulating the flavivirus lifecycle, and thereby also opens up potential new avenues for the development of antivirals targeting these conserved RNA structures. In particular, the structure we observe suggests that the plastic junction between the small hairpin and the tail of the longer stem-loop could provide a binding pocket for small molecules, for example potentially stabilizing the RNA in a conformation which hinders the conformational rearrangements critical for viral replication.

The 5'-terminal stem-loop RNA element of SARS-CoV-2 features highly dynamic structural elements that are sensitive to differences in cellular pH.

We present the nuclear magnetic resonance spectroscopy (NMR) solution structure of the 5'-terminal stem loop 5_SL1 (SL1) of the SARS-CoV-2 genome. SL1 contains two A-form helical elements and two regions with non-canonical structure, namely an apical pyrimidine-rich loop and an asymmetric internal loop with one and two nucleotides at the 5'- and 3'-terminal part of the sequence, respectively. The conformational ensemble representing the averaged solution structure of SL1 was validated using NMR residual dipolar coupling (RDC) and small-angle X-ray scattering (SAXS) data. We show that the internal loop is the major binding site for fragments of low molecular weight. This internal loop of SL1 can be stabilized by an A12-C28 interaction that promotes the transient formation of an A+•C base pair. As a consequence, the pKa of the internal loop adenosine A12 is shifted to 5.8, compared to a pKa of 3.63 of free adenosine. Furthermore, applying a recently developed pH-differential mutational profiling (PD-MaP) approach, we not only recapitulated our NMR findings of SL1 but also unveiled multiple sites potentially sensitive to pH across the 5'-UTR of SARS-CoV-2.

Structural basis of pri-let-7 recognition by human pseudouridine synthase TruB1

Let-7 was one of the first microRNAs (miRNAs) to be discovered and its expression promotes differentiation during development and function as tumor suppressors in various cancers. The maturation process of let-7 miRNA is tightly regulated by multiple RNA-binding proteins. For example, LIN28 binds to the terminal loops of the precursors of let-7 family and block their processing into mature miRNAs. Trim25 promotes the uridylation-mediated degradation of pre-let-7 modified by LIN28/TUT4. Recently, human pseudouridine synthase TruB1 has been reported to facilitate let-7 maturation by directly binding to pri-let-7 and recruiting Drosha-DGCR8 microprocessor. Through biochemical assay and structural investigation, we show that human TruB1 binds specifically the terminal loop of pri-let-7a1 at nucleotides 31–41, which folds as a small stem-loop architecture. Although TruB1 recognizes the terminal loop of pri-let-7a1 in a way similar to how E. coli TruB interacts with tRNA, a conserved KRKK motif in human and other higher eukaryotes adds an extra binding interface and strengthens the recognition of TruB1 for pri-let-7a1 through electrostatic interactions. These findings reveal the structural basis of TruB1-pri-let-7 interaction which may assists the elucidation of precise role of TruB1 in biogenesis of let-7.

No evidence of increased anti-M3 muscarinic acetylcholine receptor autoantibodies in SSA-positive connective tissue disease patients.

Anti-muscarinic type 3 receptor autoantibodies (M3R) and anti-SSA antibodies are both related to salivary secretion. The presence of M3R antibodies in Sjögren's syndrome is previously demonstrated; nevertheless, the relationship between the anti-SSA antibodies and M3R fragment antibodies, namely the N terminal, first, second, and third extracellular loops, remains to be elucidated. In this study, we analyzed the antibodies against the M3R epitopes in healthy controls and anti-SSA antibody-positive connective tissue disease patients through ELISA method. Antibodies against the first, second, and third extracellular loop (M3R211-230 ) were not increased in anti-SSA positive patients compared to healthy controls. Indeed, antibodies against the N terminal (M3R1-33 ) were found to be high in healthy controls. High levels of M3R1-33 in healthy controls are a novel original finding; further research is needed for the clinical significance. There is no significant difference between SSA-positive patients and healthy controls in terms of autoantibodies against the remainder of the linear M3R fragments.

Volterra LMS/F Based Control Algorithm for UPQC With Multi-Objective Optimized PI Controller Gains

This paper describes Volterra expansions filter with Least Mean Square (LMS) and Least Mean Fourth (LMF) control algorithm on Unified Power Quality Controller (UPQC). The adaptive models based on the LMS provide a simplified structure to approximate the necessary parameters for a particular PQ interruption. However, these algorithms are ineffective in the case of time-varying perturbations. Besides that, LMF is a comparatively better solution for multiple PQ disruptions with high computational complexity than LMS. So the LMS/Fourth (LMS/F) algorithm is a mixture of LMS and LMF to improve the error convergence quality of a given filter. The Volterra expansions filter has the advantageous characteristic of estimating fundamental components at any PQ conditions without any delay and less computational calculation. Furthermore, an optimization approach named Non-dominated Sorting Genetic Algorithm-II (NSGA-II) is used to calculate Proportional integral (PI) controller gains in the Volterra LMS/F control algorithms. The PI controller gains of dc link voltage control loop and ac terminal voltage control loop are optimized in a coordinated manner using the multi-objective NSGA-II. The applied algorithms deliver Pareto’s set of optimal global solutions. This complete algorithm is implemented through MATLAB in four-wire UPQC for mitigation of power quality problems.

Pan-Neurofascin autoimmune nodopathy - a life-threatening, but reversible neuropathy.

Autoimmune nodopathies are immune-mediated neuropathies associated with antibodies targeting the peripheral node of Ranvier. Recently, antibodies against all neurofascin-isoforms (pan-neurofascin) have been linked to a clinical phenotype distinct from previously described autoimmune nodopathies. Here, we aim at highlighting the molecular background and the red flags for diagnostic assessment and provide treatment and surveillance approaches for this new disease. Neurofascin-isoforms are located at different compartments of the node of Ranvier: Neurofascin-186 at the axonal nodal gap, and Neurofascin-155 at the terminal Schwann cell loops at the paranode. Pan-neurofascin antibodies recognize a common epitope on both isoforms and can access the node of Ranvier directly. Depending on their subclass profile, antibodies can induce direct structural disorganization and complement activation. Affected patients present with acute and immobilizing sensorimotor neuropathy, with cranial nerve involvement and long-term respiratory insufficiency. Early antibody-depleting therapy is crucial to avoid axonal damage, and remission is possible despite extended disease and high mortality. The antibody titer and serum neurofilament light chain levels can serve as biomarkers for diagnosis and therapy monitoring. Pan-neurofascin-associated autoimmune nodopathies has unique molecular and clinical features. Testing should be considered in severe and prolonged Guillain-Barré-like phenotype.

A putative wild-type or wild-type-like hairpin structure is required within 3′ untranslated region of Senecavirus A for virus replication

The 3′ untranslated region (UTR) of Senecavirus A (SVA) was predicted to harbor two hairpin structures, hairpin-I and -II. The former is composed of two internal loops, one terminal loop and three stem regions; the latter comprises one internal loop, one terminal loop and two stem regions. In this study, we constructed a total of nine SVA cDNA clones, which contained different point mutations within a stem-formed motif in the hairpin-I or -II, for rescuing replication-competent viruses. Only three mutants were successfully rescued and moreover genetically stable during at least five serial passages. Computer-aided prediction showed these three mutants bearing either a wild-type or a wild-type-like hairpin-I in their individual 3′ UTRs. Neither wild-type nor wild-type-like hairpin-I could be computationally predicted to exist in 3′ UTRs of the other six unviable “viruses”. The results suggested that the wild-type or wild-type-like hairpin-I was necessary in the 3′ UTR for SVA replication.

Origin of anomalous instability of grid‐forming converters tied to stiff grid

AbstractGrid‐forming (GFM) converter is believed to be highly promising in the future power systems, due to its ability of providing voltage and frequency support. However, some recent studies have shown that the GFM converter may suffer from instability in stiff grids, which seriously hampers its application. In this paper, the mechanism of this anomalous effect is studied by using the small‐signal stability analysis in detail. First, a detailed state‐space model of a single converter grid‐tied system is established from the first principle, and by using the participation factor analysis, the interaction between the terminal voltage loop and the power synchronization loop is identified as the major cause for the system instability. Then relying on a reduced‐order model containing only these two controls and using two classical analytical methods including the Routh criterion analysis and the Phillips– Heffron model of complex torque analysis, the origin of this anomalous instability of GFM converters tied to stiff grid coming from the negative damping provided by the terminal voltage loop is well uncovered and the critical grid strength is well predicted. In addition, these results may provide ideas for subsequent control optimization and stability improvement of GFM converters under various situations.

Delta SARS-CoV-2 s2m Structure, Dynamics, and Entropy: Consequences of the G15U Mutation.

Bioinformatic analysis of the Delta SARS-CoV-2 genome reveals a single nucleotide mutation (G15U) in the stem-loop II motif (s2m) relative to ancestral SARS-CoV-2. Despite sequence similarity, unexpected differences between SARS-CoV-2 and Delta SARS-CoV-2 s2m homodimerization experiments require the discovery of unknown structural and thermodynamic changes necessary to rationalize the data. Using our reported SARS-CoV-2 s2m model, we induced the G15U substitution and performed 3.5 microseconds of unbiased molecular dynamics simulation at 283 and 310 K. The resultant Delta s2m adopted a secondary structure consistent with our reported NMR data, resulting in significant deviations in the tertiary structure and dynamics from our SARS-CoV-2 s2m model. First, we find differences in the overall three-dimensional structure, where the characteristic 90° L-shaped kink of the SARS-CoV-2 s2m did not form in the Delta s2m resulting in a "linear" hairpin with limited bending dynamics. Delta s2m helical parameters are calculated to align closely with A-form RNA, effectively eliminating a hinge point to form the L-shape kink by correcting an upper stem defect in SARS-CoV-2 induced by a noncanonical and dynamic G:A base pair. Ultimately, the shape difference rationalizes the migration differences in reported electrophoresis experiments. Second, increased fluctuation of the Delta s2m palindromic sequence, within the terminal loop, compared to SARS-CoV-2 s2m results in an estimated increase of entropy of 6.8 kcal/mol at 310 K relative to the SARS-CoV-2 s2m. The entropic difference offers a unique perspective on why the Delta s2m homodimerizes less spontaneously, forming fewer kissing dimers and extended duplexes compared to SARS-CoV-2. In this work, both the L-shape reduction and palindromic entropic penalty provides an explanation of our reported in vitro electrophoresis homodimerization results. Ultimately, the structural, dynamical, and entropic differences between the SARS-CoV-2 s2m and Delta s2m serve to establish a foundation for future studies of the s2m function in the viral lifecycle.

Obstructed left – sided Amyand’s hernia in a toddler

Amyand’s hernia refers to an inguinal hernia which contained the vermiform appendix in the hernia sac. It is more commonly found on the right side in relation to anatomical location of the vermiform appendix and left – sided Amyand’s hernia is very rare. Herein, we report a case of left – sided Amyand’s hernia with bowel obstruction in a 1 year 4 months old boy. The left groin mass was firm and tender, with skin erythema. However, the mass could not be completed incarcerated into the abdominal cavity. His abdomen was distended with sluggish bowel sound. A diagnosis of intestinal obstruction secondary to obstructed left inguino – scrotal hernia was made, and laparotomy was performed. During laparotomy, transverse skin incision was made on the upper abdomen, and the left inguinal canal was opened in accordance with the usual inguinal hernia repair. The hernia sac contained the appendix, caecum and twisted terminal ileal loops. Small bowel resection with primary anastomosis was performed with left herniorrhaphy. Appendix was preserved due to its normal appearance. He remained well during follow up with no hernia recurrence and testicular atrophy Bangladesh Journal of Medical Science Vol. 22 No. 02 April’23 Page : 449-453

Delta variant versus ancestral SARS-CoV-2 s2m dynamics and relative entropy.

Bioinformatic analysis of the Delta SARS-CoV-2 genome reveals a single nucleotide mutation (G15U) in the stem-loop II motif (s2m) relative to ancestral SARS-CoV-2. However, the physical consequences of this change remains unexplained. Using our reported SARS-CoV-2 s2m model, we induced the G15U substitution and performed 3.5 microseconds of unbiased molecular dynamics simulation at each of 283 K and 310 K. The resultant Delta s2m adopted a secondary structure consistent with our NMR data, resulting in significant deviations in tertiary structure and dynamics from our SARS-CoV-2 s2m model. First, we find differences in three-dimensional structure, where the characteristic 90° L-shaped kink of the SARS-CoV-2 s2m never manifested in the Delta s2m (0°). Second, increased fluctuation of the Delta s2m palindromic sequence within the terminal loop results in an estimated increase of entropy of 6.82 kcal/mol at 310 K relative to the SARS-CoV-2 s2m. In this work, structural, dynamical, and entropic differences are defined between the SARS-CoV-2 s2m and Delta s2m, explaining the absence of a kissing homodimer band in our PAGE experiments and providing a foundation for future studies of s2m.

Structural and thermodynamic consequences of the G31U and U5C mutations in the SARS-CoV-2 s2m.

Within the 3’-untranslated region of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome, the stem-loop II motif (s2m) RNA element harbors two mutations (U5C and G31U) that alter the secondary structure and suspected roles in the viral life cycle compared to the SARS-CoV s2m sequence. From previously reported NMR and homodimerization experiments, the G31U mutation alone gives rise to a secondary structure and dimerization properties similar to SARS-CoV-2 s2m, while the U5C mutation was found to be more characteristic of SARS-CoV s2m. The individual effects of each mutation on the structure, dynamics, and thermodynamics have not been reported and could guide the discovery of potential functional differences of s2m in distinct viral genomes. Unbiased 3.5 microsecond molecular dynamics simulations were performed using previously reported three-dimensional atomistic coordinates based on NMR NOE assignments and the SARS-CoV s2m 1XJR crystallographic coordinates for each U5C and G31U mutation independently. Tertiary structural dependence on temperature was investigated by running simulations at 283 and 310 K to represent NMR and physiological temperatures, respectively. Principal component analysis was used to identify s2m conformational substates, and a quasiharmonic partition function was used to estimate the absolute entropy associated with each model to quantitate the structural and dynamical impact of each individual mutation. Starting with the SARS-CoV-2 s2m structure, the G31U simulation remains consistent base-pairing with our 1H NMR spectrum, while the U5C model loses the base pairing pattern. Our work addresses the structural and terminal loop entropy differences as interpretations for previously reported NMR and homodimerization experiments. Overall, a quantitative structural and dynamical insight with atomistic resolution for each U5C and G31U mutation contribution is provided for the experimentally observed deviations between SARS-CoV and SARS-CoV-2 s2m.

How AlphaFold2 Predicts Conditionally Folding Regions Annotated in an Intrinsically Disordered Protein Database, IDEAL.

AlphaFold2 (AF2) is a protein structure prediction program which provides accurate models. In addition to predicting structural domains, AF2 assigns intrinsically disordered regions (IDRs) by identifying regions with low prediction reliability (pLDDT). Some regions in IDRs undergo disorder-to-order transition upon binding the interaction partner. Here we assessed model structures of AF2 based on the annotations in IDEAL, in which segments with disorder-to-order transition have been collected as Protean Segments (ProSs). We non-redundantly selected ProSs from IDEAL and classified them based on the root mean square deviation to the corresponding region of AF2 models. Statistical analysis identified 11 structural and sequential features, possibly contributing toward the prediction of ProS structures. These features were categorized into two groups: one that contained pLDDT and the other that contained normalized radius of gyration. The typical ProS structures in the former group comprise a long α helix or a whole or part of the structural domain and those in the latter group comprise a short α helix with terminal loops.

Iron-dependent post transcriptional control of mitochondrial aconitase expression.

Iron regulatory proteins (IRPs) control the translation of animal cell mRNAs encoding proteins with diverse roles. This includes the iron storage protein ferritin and the tricarboxylic cycle (TCA) enzyme mitochondrial aconitase (ACO2) through iron-dependent binding of IRP to the iron responsive element (IRE) in the 5' untranslated region (UTR). To further elucidate the mechanisms allowing IRPs to control translation of 5' IRE-containing mRNA differentially, we focused on Aco2 mRNA, which is weakly controlled versus the ferritins. Rat liver contains two classes of Aco2 mRNAs, with and without an IRE, due to alterations in the transcription start site. Structural analysis showed that the Aco2 IRE adopts the canonical IRE structure but lacks the dynamic internal loop/bulge five base pairs 5' of the CAGUG(U/C) terminal loop in the ferritin IREs. Unlike ferritin mRNAs, the Aco2 IRE lacks an extensive base-paired flanking region. Using a full-length Aco2 mRNA expression construct, iron controlled ACO2 expression in an IRE-dependent and IRE-independent manner, the latter of which was eliminated with the ACO23C3S mutant that cannot bind the FeS cluster. Iron regulation of ACO23C3S encoded by the full-length mRNA was completely IRE-dependent. Replacement of the Aco23C3S 5' UTR with the Fth1 IRE with base-paired flanking sequences substantially improved iron responsiveness, as did fusing of the Fth1 base-paired flanking sequences to the native IRE in the Aco3C3S construct. Our studies further define the mechanisms underlying the IRP-dependent translational regulatory hierarchy and reveal that Aco2 mRNA species lacking the IRE contribute to the expression of this TCA cycle enzyme.

Structural, Dynamical, and Entropic Differences between SARS-CoV and SARS-CoV-2 s2m Elements Using Molecular Dynamics Simulations.

The functional role of the highly conserved stem-loop II motif (s2m) in SARS-CoV and SARS-CoV-2 in the viral lifecycle remains enigmatic and an intense area of research. Structure and dynamics of the s2m are key to establishing a structure–function connection, yet a full set of atomistic resolution coordinates is not available for SARS-CoV-2 s2m. Our work constructs three-dimensional coordinates consistent with NMR solution phase data for SARS-CoV-2 s2m and provides a comparative analysis with its counterpart SARS-CoV s2m. We employed initial coordinates based on PDB ID 1XJR for SARS-CoV s2m and two models for SARS-CoV-2 s2m: one based on 1XJR in which we introduced the mutations present in SARS-CoV-2 s2m and the second based on the available SARS-CoV-2 NMR NOE data supplemented with knowledge-based methods. For each of the three systems, 3.5 μs molecular dynamics simulations were used to sample the structure and dynamics, and principal component analysis (PCA) reduced the ensembles to hierarchal conformational substates for detailed analysis. Dilute solution simulations of SARS-CoV s2m demonstrate that the GNRA-like terminal pentaloop is rigidly defined by base stacking uniquely positioned for possible kissing dimer formation. However, the SARS-CoV-2 s2m simulation did not retain the reported crystallographic SARS-CoV motifs and the terminal loop expands to a highly dynamic “nonaloop.” Increased flexibility and structural disorganization are observed for the larger terminal loop, where an entropic penalty is computed to explain the experimentally observed reduction in kissing complex formation. Overall, both SARS-CoV and SARS-CoV-2 s2m elements have a similarly pronounced L-shape due to different motif interactions. Our study establishes the atomistic three-dimensional structure and uncovers dynamic differences that arise from s2m sequence changes, which sets the stage for the interrogation of different mechanistic pathways of suspected biological function.

Interaction Mechanism Analysis and Additional Damping Control for Hybrid Multi-Infeed HVDC System

With the emergence of the hybrid multi-infeed high voltage direct current (HMIDC) system, there is complex dynamic interaction between the AC/DC system and converter stations, while the interaction mechanism of the HMIDC system is unclear. For this purpose, this paper establishes the HMIDC linearized model based on the motion equation concept. Then, after decoupling and reconstructing the interaction path of the HMIDC system, the damping coefficients are derived. The AC system strength and voltage source converter (VSC) terminal voltage controller parameters are selected to analyze the impacts on damping coefficients, and the interaction mechanism of the HMIDC system is analyzed. In order to solve the potential instability issue, an additional damping controller in the VSC terminal voltage loop is proposed to enhance the voltage stability of HMIDC system, which is realized by adjusting the reactive power/amplitude coupling damping coefficient. Finally, the RT-LAB-based hardware-in-the-loop experiments verify the accuracy of the interaction mechanism analysis of the HMIDC system and the effectiveness of the proposed controller.

Regulated dicing of pre-mir-144 via reshaping of its terminal loop.

Although the route to generate microRNAs (miRNAs) is often depicted as a linear series of sequential and constitutive cleavages, we now appreciate multiple alternative pathways as well as diverse strategies to modulate their processing and function. Here, we identify an unusually profound regulatory role of conserved loop sequences in vertebrate pre-mir-144, which are essential for its cleavage by the Dicer RNase III enzyme in human and zebrafish models. Our data indicate that pre-mir-144 dicing is positively regulated via its terminal loop, and involves the ILF3 complex (NF90 and its partner NF45/ILF2). We provide further evidence that this regulatory switch involves reshaping of the pre-mir-144 apical loop into a structure that is appropriate for Dicer cleavage. In light of our recent findings that mir-144 promotes the nuclear biogenesis of its neighbor mir-451, these data extend the complex hierarchy of nuclear and cytoplasmic regulatory events that can control the maturation of clustered miRNAs.

Conformational Effects of a Cancer-Linked Mutation in Pri-miR-30c RNA

MicroRNAs (miRNAs) are small, noncoding RNAs that mediate post-transcriptional downregulation of specific target genes. These transcripts are the products of a two-step processing pathway; primary miRNAs (pri-miRNAs) are processed by Drosha into individual precursor miRNA (pre-miRNA) hairpins, which are subsequently processed by Dicer into mature miRNAs. Single nucleotide polymorphisms (SNPs) that occur in pri-miRNAs, pre-miRNAs and mature miRNAs have been shown to affect the processing of specific target genes by modulating Drosha and Dicer processing or interactions with RNA binding proteins (RBPs). Using NMR and single-molecule optical tweezer experiments, we have investigated the conformational effects of a cancer-linked G/A mutation in the terminal loop of pri-miR-30c RNA, and how this influences binding by the SRSF3 and hnRNP A1 RBPs, which are implicated in its processing. Our results reveal that the wildtype and G/A variant pri-miR-30c RNAs adopt very similar elongated stem-loop structures, both of which are bound by SRSF3. However, while both wildtype and G/A pri-miR-30c RNAs can form dimeric kissing hairpin structures, the G to A mutation results in partial destabilization of the dimer in the variant transcript. This promotes recognition and binding by hnRNP A1, an RBP that enhances pri-miR-30c processing. Our data provide structural insight into the conformational effects of a G/A mutation in pri-miR-30c RNA and how this could affect processing and promote cancer.

Molecular Dissection of a Conserved Cluster of miRNAs Identifies Critical Structural Determinants That Mediate Differential Processing.

Differential processing is a hallmark of clustered microRNAs (miRNAs) and the role of position and order of miRNAs in a cluster together with the contribution of stem-base and terminal loops has not been explored extensively within the context of a polycistronic transcript. To elucidate the structural attributes of a polycistronic transcript that contribute towards the differences in efficiencies of processing of the co-transcribed miRNAs, we constructed a series of chimeric variants of Drosophila let-7-Complex that encodes three evolutionary conserved and differentially expressed miRNAs (miR-100, let-7 and miR-125) and examined the expression and biological activity of the encoded miRNAs. The kinetic effects of Drosha and Dicer processing on the chimeric precursors were examined by in vitro processing assays. Our results highlight the importance of stem-base and terminal loop sequences in differential expression of polycistronic miRNAs and provide evidence that processing of a particular miRNA in a polycistronic transcript is in part determined by the kinetics of processing of adjacent miRNAs in the same cluster. Overall, this analysis provides specific guidelines for achieving differential expression of a particular miRNA in a cluster by structurally induced changes in primary miRNA (pri-miRNA) sequences.

Crystal structure of Acetyl-CoA carboxylase (AccB) from Streptomyces antibioticus and insights into the substrate-binding through in silico mutagenesis and biophysical investigations

Acetyl-CoA carboxylase (ACC) is crucial for polyketides biosynthesis and acts as an essential metabolic checkpoint. It is also an attractive drug target against obesity, cancer, microbial infections, and diabetes. However, the lack of knowledge, particularly sequence-structure function relationship to narrate ligand-enzyme binding, has hindered the progress of ACC-specific therapeutics and unnatural “natural” polyketides. Structural characterization of such enzymes will boost the opportunity to understand the substrate binding, designing new inhibitors and information regarding the molecular rules which control the substrate specificity of ACCs. To understand the substrate specificity, we determined the crystal structure of AccB (Carboxyl-transferase, CT) from Streptomyces antibioticus with a resolution of 2.3 Å and molecular modeling approaches were employed to unveil the molecular mechanism of acetyl-CoA recognition and processing. The CT domain of S. antibioticus shares a similar structural organization with the previous structures and the two steps reaction was confirmed by enzymatic assay. Furthermore, to reveal the key hotspots required for the substrate recognition and processing, in silico mutagenesis validated only three key residues (V223, Q346, and Q514) that help in the fixation of the substrate. Moreover, we also presented atomic level knowledge on the mechanism of the substrate binding, which unveiled the terminal loop (500–514) function as an opening and closing switch and pushes the substrate inside the cavity for stable binding. A significant decline in the hydrogen bonding half-life was observed upon the alanine substitution. Consequently, the presented structural data highlighted the potential key interacting residues for substrate recognition and will also help to re-design ACCs active site for proficient substrate specificity to produce diverse polyketides.