The life cycle of Baculoviridae family insect viruses depends on the viral protein kinase, PK-1, to phosphorylate the regulatory protein, p6.9, to induce baculoviral genome release. Here, we report ...the crystal structure of Cydia pomenella granulovirus PK-1, which, owing to its likely ancestral origin among host cell AGC kinases, exhibits a eukaryotic protein kinase fold. PK-1 occurs as a rigid dimer, where an antiparallel arrangement of the αC helices at the dimer core stabilizes PK-1 in a closed, active conformation. Dimerization is facilitated by C-lobe:C-lobe and N-lobe:N-lobe interactions between protomers, including the domain-swapping of an N-terminal helix that crowns a contiguous β-sheet formed by the two N-lobes. PK-1 retains a dimeric conformation in solution, which is crucial for catalytic activity. Our studies raise the prospect that parallel, side-to-side dimeric arrangements that lock kinase domains in a catalytically-active conformation could function more broadly as a regulatory mechanism among eukaryotic protein kinases.
Abstract
Across the globe, 2-3% of humans carry the
p.Ser132Pro
single nucleotide polymorphism in
MLKL
, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. ...Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKL
S132P
in biological membranes and MLKL
S132P
overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent
Mlkl S131P
mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-β induced death in non-hematopoietic cells. In vivo,
Mlkl
S131P
homozygosity reduces the capacity to clear
Salmonella
from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the
MLKL S132P
polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.
TRAPs: the 'elevator-with-an-operator' mechanism Davies, James S; Currie, Michael J; Dobson, Renwick C J ...
Trends in biochemical sciences (Amsterdam. Regular ed.),
02/2024, Letnik:
49, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Tripartite ATP-independent periplasmic (TRAP) transporters are nutrient-uptake systems found in bacteria and archaea. These evolutionary divergent transporter systems couple a substrate-binding ...protein (SBP) to an elevator-type secondary transporter, which is a first-of-its-kind mechanism of transport. Here, we highlight breakthrough TRAP transporter structures and recent functional data that probe the mechanism of transport. Furthermore, we discuss recent structural and biophysical studies of the ion transporter superfamily (ITS) members and highlight mechanistic principles that are relevant for further exploration of the TRAP transporter system.
Abstract
The necroptosis pathway is a lytic, pro-inflammatory mode of cell death that is widely implicated in human disease, including renal, pulmonary, gut and skin inflammatory pathologies. The ...precise mechanism of the terminal steps in the pathway, where the RIPK3 kinase phosphorylates and triggers a conformation change and oligomerization of the terminal pathway effector, MLKL, are only emerging. Here, we structurally identify RIPK3-mediated phosphorylation of the human MLKL activation loop as a cue for MLKL pseudokinase domain dimerization. MLKL pseudokinase domain dimerization subsequently drives formation of elongated homotetramers. Negative stain electron microscopy and modelling support nucleation of the MLKL tetramer assembly by a central coiled coil formed by the extended, ~80 Å brace helix that connects the pseudokinase and executioner four-helix bundle domains. Mutational data assert MLKL tetramerization as an essential prerequisite step to enable the release and reorganization of four-helix bundle domains for membrane permeabilization and cell death.
Human pathogenic and commensal bacteria have evolved the ability to scavenge host‐derived sialic acids and subsequently degrade them as a source of nutrition. Expression of the Escherichia coli yjhBC ...operon is controlled by the repressor protein nanR, which regulates the core machinery responsible for the import and catabolic processing of sialic acid. The role of the yjhBC encoded proteins is not known—here, we demonstrate that the enzyme YjhC is an oxidoreductase/dehydrogenase involved in bacterial sialic acid degradation. First, we demonstrate in vivo using knockout experiments that YjhC is broadly involved in carbohydrate metabolism, including that of N‐acetyl‐d‐glucosamine, N‐acetyl‐d‐galactosamine and N‐acetylneuraminic acid. Differential scanning fluorimetry demonstrates that YjhC binds N‐acetylneuraminic acid and its lactone variant, along with NAD(H), which is consistent with its role as an oxidoreductase. Next, we solved the crystal structure of YjhC in complex with the NAD(H) cofactor to 1.35 Å resolution. The protein fold belongs to the Gfo/Idh/MocA protein family. The dimeric assembly observed in the crystal form is confirmed through solution studies. Ensemble refinement reveals a flexible loop region that may play a key role during catalysis, providing essential contacts to stabilize the substrate—a unique feature to YjhC among closely related structures. Guided by the structure, in silico docking experiments support the binding of sialic acid and several common derivatives in the binding pocket, which has an overall positive charge distribution. Taken together, our results verify the role of YjhC as a bona fide oxidoreductase/dehydrogenase and provide the first evidence to support its involvement in sialic acid metabolism.
Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed ...to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL's N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.
Thioamides are single atom substitutions of the peptide bond that serve as versatile probes of protein structure. Effective use of thioamides requires a robust understanding of the impact that the ...substitution has on a protein of interest. However, the thermodynamic effects of thioamide incorporation have only been studied in small structural motifs, and their influence on secondary structure in the context of full-length proteins is not known. Here we describe a comprehensive survey of thioamide substitutions in three benchmark protein systems (calmodulin, the B1 domain of protein G, and collagen) featuring the most prevalent secondary structure motifs: α-helix, β-sheet, and polyproline type II helix. We find that in most cases, effects on thermostability can be understood in terms of the positioning and local environment of the thioamide relative to proximal structural elements and hydrogen bonding networks. These observations set the stage for the rational design of thioamide substituted proteins with predictable stabilities.
There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been ...proposed to use a deprotonation–reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the β-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.
In environments where glucose is limited, some pathogenic bacteria metabolize host-derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial ...sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, ORF, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding EXGH motif is altered to EXGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterization of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand–free, N-acetylmannosamine–bound and substrate analog GlcNAc–bound crystal structures (2.33, 2.20, and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the EXGY motif reveals that the tyrosine binds to the N-acetyl group to select for the “boat” conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterization provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.
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