Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of ...anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.
Transient receptor potential vanilloid channel 3 (TRPV3), a member of the thermosensitive TRP (thermoTRPV) channels, is activated by warm temperatures and serves as a key regulator of normal skin ...physiology through the release of pro-inflammatory messengers. Mutations in trpv3 have been identified as the cause of the congenital skin disorder, Olmsted syndrome. Unlike other members of the thermoTRPV channel family, TRPV3 sensitizes upon repeated stimulation, yet a lack of structural information about the channel precludes a molecular-level understanding of TRPV3 sensitization and gating. Here, we present the cryo-electron microscopy structures of apo and sensitized human TRPV3, as well as several structures of TRPV3 in the presence of the common thermoTRPV agonist 2-aminoethoxydiphenyl borate (2-APB). Our results show α-to-π-helix transitions in the S6 during sensitization, and suggest a critical role for the S4-S5 linker π-helix during ligand-dependent gating.
Human mitochondrial gene expression relies on the specific recognition and aminoacylation of mitochondrial tRNAs (mtRNAs) by nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs). ...Despite their essential role in cellular energy homeostasis, strong mutation pressure and genetic drift have led to an unparalleled sequence erosion of animal mtRNAs. The structural and functional consequences of this erosion are not understood. Here, we present cryo-EM structures of the human mitochondrial seryl-tRNA synthetase (mSerRS) in complex with mtRNA
. These structures reveal a unique mechanism of substrate recognition and aminoacylation. The mtRNA
is highly degenerated, having lost the entire D-arm, tertiary core, and stable L-shaped fold that define canonical tRNAs. Instead, mtRNA
evolved unique structural innovations, including a radically altered T-arm topology that serves as critical identity determinant in an unusual shape-selective readout mechanism by mSerRS. Our results provide a molecular framework to understand the principles of mito-nuclear co-evolution and specialized mechanisms of tRNA recognition in mammalian mitochondrial gene expression.
Animal mitochondrial gene expression relies on specific interactions between nuclear-encoded aminoacyl-tRNA synthetases and mitochondria-encoded tRNAs. Their evolution involves an antagonistic ...interplay between strong mutation pressure on mtRNAs and selection pressure to maintain their essential function. To understand the molecular consequences of this interplay, we analyze the human mitochondrial serylation system, in which one synthetase charges two highly divergent mtRNA
isoacceptors. We present the cryo-EM structure of human mSerRS in complex with mtRNA
, and perform a structural and functional comparison with the mSerRS-mtRNA
complex. We find that despite their common function, mtRNA
and mtRNA
show no constrain to converge on shared structural or sequence identity motifs for recognition by mSerRS. Instead, mSerRS evolved a bimodal readout mechanism, whereby a single protein surface recognizes degenerate identity features specific to each mtRNA
. Our results show how the mutational erosion of mtRNAs drove a remarkable innovation of intermolecular specificity rules, with multiple evolutionary pathways leading to functionally equivalent outcomes.
A number of human malignancies exhibit sustained stimulation, mutation, or gene amplification of the receptor tyrosine kinase human mesenchymal-epithelial transition factor (c-Met). ARQ 197 is a ...clinically advanced, selective, orally bioavailable, and well tolerated c-Met inhibitor, currently in Phase 3 clinical testing in non-small cell lung cancer patients. Herein, we describe the molecular and structural basis by which ARQ 197 selectively targets c-Met. Through our analysis we reveal a previously undisclosed, novel inhibitory mechanism that utilizes distinct regulatory elements of the c-Met kinase. The structure of ARQ 197 in complex with the c-Met kinase domain shows that the inhibitor binds a conformation that is distinct from published kinase structures. ARQ 197 inhibits c-Met autophosphorylation and is highly selective for the inactive or unphosphorylated form of c-Met. Through our analysis of the interplay between the regulatory and catalytic residues of c-Met, and by comparison between the autoinhibited canonical conformation of c-Met bound by ARQ 197 to previously described kinase domains of type III receptor tyrosine kinases, we believe this to be the basis of a powerful new in silico approach for the design of similar inhibitors for other protein kinases of therapeutic interest.
Membrane transporters move substrates across the membrane by alternating access of their binding sites between the opposite sides of the membrane. An emerging model of this process is the elevator ...mechanism, in which a substrate-binding transport domain moves a large distance across the membrane. This mechanism has been characterized by a transition between two states, but the conformational path that leads to the transition is not yet known, largely because the available structural information has been limited to the two end states. Here we present crystal structures of the inward-facing, intermediate, and outward-facing states of a concentrative nucleoside transporter from Neisseria wadsworthii. Notably, we determined the structures of multiple intermediate conformations, in which the transport domain is captured halfway through its elevator motion. Our structures present a trajectory of the conformational transition in the elevator model, revealing multiple intermediate steps and state-dependent conformational changes within the transport domain that are associated with the elevator-like motion.
The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signalling. The transient receptor potential mucolipin (TRPML) channel family ...belongs to the TRP superfamily and is composed of three members: TRPML1-TRPML3. TRPMLs are the major Ca
-permeable channels on late endosomes and lysosomes (LEL). They regulate the release of Ca
from organelles, which is important for various physiological processes, including organelle trafficking and fusion. Loss-of-function mutations in the MCOLN1 gene, which encodes TRPML1, cause the neurodegenerative lysosomal storage disorder mucolipidosis type IV, and a gain-of-function mutation (Ala419Pro) in TRPML3 gives rise to the varitint-waddler (Va) mouse phenotype. Notably, TRPML channels are activated by the low-abundance and LEL-enriched signalling lipid phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P
), whereas other phosphoinositides such as PtdIns(4,5)P
, which is enriched in plasma membranes, inhibit TRPMLs. Conserved basic residues at the N terminus of the channel are important for activation by PtdIns(3,5)P
and inhibition by PtdIns(4,5)P
. However, owing to a lack of structural information, the mechanism by which TRPML channels recognize PtdIns(3,5)P
and increase their Ca
conductance remains unclear. Here we present the cryo-electron microscopy (cryo-EM) structure of a full-length TRPML3 channel from the common marmoset (Callithrix jacchus) at an overall resolution of 2.9 Å. Our structure reveals not only the molecular basis of ion conduction but also the unique architecture of TRPMLs, wherein the voltage sensor-like domain is linked to the pore via a cytosolic domain that we term the mucolipin domain. Combined with functional studies, these data suggest that the mucolipin domain is responsible for PtdIns(3,5)P
binding and subsequent channel activation, and that it acts as a 'gating pulley' for lipid-dependent TRPML gating.
The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signaling. The mucolipin transient receptor potential (TRPML) channel family belongs ...to the TRP superfamily
1
,
2
and is composed of three members, TRPML1-3. TRPMLs are the major Ca
2+
-permeable channels on late endosomes and lysosomes (LEL). They regulate organelle Ca
2+
releases important for various physiological processes, including organelle trafficking and fusion
3
. Loss-of-function mutations in the TRPML1 gene cause the neurodegenerative lysosomal storage disorder mucolipidosis IV (ML-IV), and a gain-of-function mutation in TRPML3 (Ala419Pro) gives rise to the Varitint-Waddler (
Va
) mouse phenotype
4
–
6
. Notably, TRPMLs are activated by the low-abundance and LEL-enriched signaling lipid PI(3,5)P
2
, while other phosphoinositides such as PI(4,5)P
2
, enriched in plasma membranes, inhibit TRPMLs
7
,
8
. Conserved basic residues at the N-terminus of the channels are important for PI(3,5)P
2
activation and PI(4,5)P
2
inhibition
8
. However, due to a lack of structural information, the mechanism by which TRPML channels recognize PI(3,5)P
2
and increase its Ca
2+
conductance remains elusive. Here we present the cryo-electron microscopy (cryo-EM) structure of a full-length TRPML3, at an average resolution of 2.9 Å. Our structure reveals not only the molecular basis of ion conduction but also the unique architecture of TRPMLs, wherein the voltage sensor-like domain is linked to the pore via a cytosolic domain we term the “mucolipin domain” (MLD). Combined with functional studies, we suggest that the MLD is responsible for PI(3,5)P
2
binding and subsequent channel activation, and that it acts as a ‘gating pulley’ for lipid-dependent TRPML gating.
Nucleoside transport is required for the salvage pathway of DNA and RNA, provides a transport route for nucleoside-like drugs and regulates adenosine signaling. The eukaryotic cell possesses two ...evolutionarily unrelated protein families of nucleoside transporters, the concentrative nucleoside transporters and the equilibrative nucleoside transporters. We aimed to elucidate their transport mechanisms using X-ray crystallography and biochemical assays. We solved multiple crystal structures of CNT along its transport cycle at 3.45-4.2 Å, which provide novel insights into the elevator-type transport mechanism of secondary active transporters.
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