Structural insights as to how iron-loaded siderophores are imported into Mycobacterium tuberculosis by IrtAB

The ATP-binding cassette (ABC) transporter, IrtAB, plays a vital role in the replication and viability of Mycobacterium tuberculosis (Mtb), where its function is to import iron-loaded siderophores. Unusually, it adopts the typical fold of canonical ABC exporters. Here, we report the structure of unliganded Mtb IrtAB and its structure in complex with ATP, ADP, and an ATP analogue (AMP-PNP) at resolutions from 2.9 to 3.5 Å. In the ATP-bound state, the two nucleotide-binding domains (NBDs) form a "head-to-tail" dimer, but IrtAB has an unexpectedly occluded conformation, with the inner core forming a large hydrophilic cavity of about 4600 Å3. Comparison of the structure of the transporter in inward-facing and occluded conformations reveals that the NBD and the intracellular helical region of transmembrane domain (TMD) have an asymmetric allosteric mechanism when ATP binding/hydrolysis such that the one exhibits rigid-body rotation and the other moves in a concerted response as a rigid body. This study provides a molecular basis for the ATP-driven conformational changes that occur in IrtAB and an explanation as to how iron-loaded siderophores are imported into Mtb by IrtAB.

Variant Type X91+ Chronic Granulomatous Disease: Clinical and Molecular Characterization in a Chinese Cohort

Purpose: We aimed to report the clinical and immunological characteristics of variant type X91+ CGD in a Chinese cohort.Methods: The clinical manifestations and immunological phenotypes of X91+ CGD patients were collected. Dihydrorhodamine (DHR) analysis was performed to evaluate neutrophil function. Gp91phox protein expression was determined by flow cytometry-based extracellular staining with the monoclonal antibody (mAb) 7D5. Results: X91+CGD patients accounted for 8% (7/85) of all patients with GCD. The median onset age in the 7 X91+ CGD patients was 4 months. Six patients received the same BCG vaccine strain, and three had probable BCG infections. Moreover, 4 patients were highly suspected of having Mycobacterium tuberculosis infection. Recurrent infections of the lungs and soft tissues (3/7) were the most common symptoms. Two patients had noninfectious recurrent oral ulcers and received interferon gamma (IFN-γ) treatment afterward. In our cohort, the stimulation index (SI) of the 7 X91+ CGD patients ranged widely from 1.9 to 67.5, while the SI ranged from 1.2 to 35.7 in patents with X910 CGD. The level of SI between these two groups was statistically significant (P<0.05). CYBB mutations associated with X91+CGD were usually located in or near the FAD and NADPH binding domains. Three new X91+ CGD related mutations (c.1462-2 A>T, c.1243C>T and c.925G>A) were identified. Conclusions: Variant type X91+ CGD may have varied severities of clinical manifestations. Moreover, the laboratory findings of X91+ CGD could present with a moderate neutrophil stimulation index. We should deepen our understanding of the X91+ variant CGD to prevent missed diagnosis.

Control of bacterial anti-phage signaling by a WYL domain transcription factor

Bacteria use diverse immune systems to defend themselves from ubiquitous viruses termed bacteriophages (phages). Many anti-phage systems function by abortive infection to kill a phage-infected cell, raising the question of how these systems are regulated to avoid activation and cell killing outside the context of infection. Here, we identify a transcription factor associated with the widespread CBASS bacterial immune system, that we term CapW. CapW forms a homodimer and binds a palindromic DNA sequence in the CBASS promoter region. Two crystal structures of CapW reveal how the protein switches from a DNA binding-competent state to a ligand-bound state that cannot bind DNA due to misalignment of dimer-related DNA binding domains. We show that CapW strongly represses CBASS gene expression in uninfected cells, and that CapW disruption likely results in toxicity due to uncontrolled CBASS expression. Our results parallel recent findings with BrxR, a transcription factor associated with the BREX anti-phage system, and suggest that CapW and BrxR are the founding members of a family of universal anti-phage signaling proteins.

Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains

Viral spillover from animal reservoirs can trigger public health crises and cripple the world economy. Knowing which viruses are primed for zoonotic transmission can focus surveillance efforts and mitigation strategies for future pandemics. Successful engagement of receptor protein orthologs is necessary during cross-species transmission. The clade 1 sarbecoviruses including SARS-CoV and SARS-CoV-2 enter cells via engagement of ACE2, while the receptor for clade 2 and clade 3 remains largely uncharacterized. We developed a mixed cell pseudotyped virus infection assay to determine whether various clade 2 and 3 sarbecovirus spike proteins can enter HEK 293T cells expressing human or Rhinolophus horseshoe bat ACE2 proteins. The receptor binding domains from BtKY72 and Khosta-2 used human ACE2 for entry, while BtKY72 and Khosta-1 exhibited widespread use of diverse rhinolophid ACE2s. A lysine at ACE2 position 31 appeared to be a major determinant of the inability of these RBDs to use a certain ACE2 sequence. The ACE2 protein from R. alcyone engaged all known clade 3 and clade 1 receptor binding domains. We observed little use of Rhinolophus ACE2 orthologs by the clade 2 viruses, supporting the likely use of a separate, unknown receptor. Our results suggest that clade 3 sarbecoviruses from Africa and Europe use Rhinolophus ACE2 for entry, and their spike proteins appear primed to contribute to zoonosis under the right conditions.

Structure and ion-release mechanism of PIB-4-type ATPases

Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here we present structures and complementary functional analyses of an archetypal PIB‑4‑ATPase, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy metal binding domains, and provides fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release and as an internal counter ion of an invariant histidine. We also establish that the turn-over of PIB‑ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in e.g. drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.

Computational Approaches to Investigate and Design Lipid-binding Domains for Membrane Biosensing

Association of proteins with cellular membranes is critical for signaling and membrane trafficking processes. Many peripheral lipid-binding domains have been identified in the last few decades and have been investigated for their specific lipid-sensing properties using traditional in vivo and in vitro studies. However, several knowledge gaps remain owing to intrinsic limitations of these methodologies. Thus, novel approaches are necessary to further our understanding in lipid–protein biology. This review briefly discusses lipid-binding domains that act as specific lipid biosensors and provides a broad perspective on the computational approaches such as molecular dynamics (MD) simulations and machine learning (ML)-based techniques that can be used to study protein–membrane interactions. We also highlight the need for de novo design of proteins that elicit specific lipid-binding properties.

The Human ATP-Binding Cassette (ABC) Transporter Superfamily

The ATP-binding cassette (ABC) transporter superfamily comprises membrane proteins that efflux various substrates across extra- and intra-cellular membranes. Mutations in ABC genes cause 21 human disorders or phenotypes with Mendelian inheritance, including cystic fibrosis, adrenoleukodystrophy, retinal degeneration, cholesterol, and bile transport defects. Common polymorphisms and rare variants in ABC genes are associated with several complex phenotypes such as gout, gallstones, and cholesterol levels. Overexpression or amplification of specific drug efflux genes contributes to chemotherapy multidrug resistance. Conservation of the ATP-binding domains of ABC transporters defines the superfamily members, and phylogenetic analysis groups the 48 human ABC transporters into seven distinct subfamilies. While the conservation of ABC genes across most vertebrate species is high, there is also considerable gene duplication, deletion, and evolutionary diversification.

Relationship between monomer packing and receptor binding domain pocket status in the spike trimer of SARS-CoV-2 variants

Existence of a SARS-CoV-2 spike protein trimer form with closer packing between monomers when receptor binding domains (RBDs) are all down, locked as opposed to closed, has been associated with linoleic acid (LA) binding at neutral pH, or can occur at acidic pH in the absence of LA binding. The relationship between degree of closure of the LA binding pocket of the RBD, and monomer burial in the trimer, is examined for a range of spike protein structures, including those with D614G mutation, and that of the Delta variant (which also carries D614G). Some spike protein structures with this aspartic acid mutation show monomer packing approaching that of the locked form (at neutral pH, without LA binding) for two segments, a third (around the RBD) remains less closely packed. Analysis of other coronavirus RBD structures suggests that mutation of the RBD in spike protein of the Omicron variant could lead to LA binding pocket changes. It is proposed that these changes could lead to one of two consequences for the Omicron variant spike protein (which also has the D614G mutation), at neutral pH and without LA binding, either easier access to a locked form throughout that leads to cooperative transitions between all RBD down and all RBD up, or maintenance of a spike trimer with locked characteristics C-terminal to the RBD at the same time as the RBD is free to transit between down and up states. The situation may also be impacted by spike protein charge mutations in the Omicron lineage that alter pH-dependence around the RBD, in a similar way to the changes induced elsewhere by D614G.