Enzymology
Cooperative dynamics across distinct structural elements regulate PTP1B activity
Protein-tyrosine phosphatase 1B (PTP1B) is the canonical enzyme for investigating how distinct structural elements influence enzyme catalytic activity. Although it is recognized that dynamics are essential for PTP1B function, the data collected thus far have not resolved whether distinct elements are dynamically coordinated or, alternatively, whether they fulfill their respective functions independently. To answer this question, we performed a comprehensive 13C-methyl relaxation study of Ile, Leu, and Val (ILV) residues of PTP1B, which, because of its substantially increased sensitivity, provides a comprehensive understanding of the influence of protein motions on different time scales for enzyme function.Cryo-EM reveals the architecture of the dimeric cytochrome P450 CYP102A1 enzyme and conformational changes required for redox partner recognition
Cytochrome P450 family 102 subfamily A member 1 (CYP102A1) is a self-sufficient flavohemeprotein and a highly active bacterial enzyme capable of fatty acid hydroxylation at a >3,000 min−1 turnover rate. The CYP102A1 architecture has been postulated to be responsible for its extraordinary catalytic prowess. However, the structure of a functional full-length CYP102A1 enzyme remains to be determined. Herein, we used a cryo-EM single-particle approach, revealing that full-length CYP102A1 forms a homodimer in which both the heme and FAD domains contact each other.Interdomain communication in the phosphatidylcholine regulatory enzyme, CCTα, relies on a modular αE helix
phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme in phosphatidylcholine (PC) synthesis, is an amphitropic enzyme that regulates PC homeostasis. Recent work has suggested that CCTα activation by binding to a PC-deficient membrane involves conformational transitions in a helix pair (αE) that, along with a short linker of unknown structure (J segment), bridges the catalytic domains of the CCTα dimer to the membrane-binding (M) domains. In the soluble, inactive form, the αE helices are constrained into unbroken helices by contacts with two auto-inhibitory (AI) helices from domain M.Tri-arginine exosite patch of caspase-6 recruits substrates for hydrolysis
Caspases are cysteine–aspartic proteases involved in the regulation of programmed cell death (apoptosis) and a number of other biological processes. Despite overall similarities in structure and active-site composition, caspases show striking selectivity for particular protein substrates. Exosites are emerging as one of the mechanisms by which caspases can recruit, engage, and orient these substrates for proper hydrolysis. Following computational analyses and database searches for candidate exosites, we utilized site-directed mutagenesis to identify a new exosite in caspase-6 at the hinge between the disordered N-terminal domain (NTD), residues 23–45, and core of the caspase-6 structure.Electrostatic interactions between middle domain motif-1 and the AAA1 module of the bacterial ClpB chaperone are essential for protein disaggregation
ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), ClpB forms a hexameric ring structure, with each protomer containing two AAA+ modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2.An auto-inhibitory helix in CTP:phosphocholine cytidylyltransferase hijacks the catalytic residue and constrains a pliable, domain-bridging helix pair
The activity of CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme in phosphatidylcholine synthesis, is regulated by reversible interactions of a lipid-inducible amphipathic helix (domain M) with membrane phospholipids. When dissociated from membranes, a portion of the M domain functions as an auto-inhibitory (AI) element to suppress catalysis. The AI helix from each subunit binds to a pair of α helices (αE) that extend from the base of the catalytic dimer to create a four-helix bundle.The cell division protein MinD from Pseudomonas aeruginosa dominates the assembly of the MinC–MinD copolymers
Cell division of rod-shaped bacteria requires the Z ring, a ring of FtsZ filaments associated with the inner-membrane wall. The MinCDE proteins help localize the Z ring to the center of the Escherichia coli cell. MinC, which inhibits Z-ring assembly, is a passenger on MinD. Previous studies have shown that MinC–MinD from E. coli and Aquifex aeolicus assemble in vitro into extended filaments with a 1:1 stoichiometry. However, a recent study has raised questions about the function of the MinC–MinD copolymer in vivo, because its assembly appears to require a high concentration of these two proteins and has a long lag time, and its blockade does not affect in vivo activities.The metal chaperone Atox1 regulates the activity of the human copper transporter ATP7B by modulating domain dynamics
The human transporter ATP7B delivers copper to the biosynthetic pathways and maintains copper homeostasis in the liver. Mutations in ATP7B cause the potentially fatal hepatoneurological disorder Wilson disease. The activity and intracellular localization of ATP7B are regulated by copper, but the molecular mechanism of this regulation is largely unknown. We show that the copper chaperone Atox1, which delivers copper to ATP7B, and the group of the first three metal-binding domains (MBD1–3) are central to the activity regulation of ATP7B.The role of the Met20 loop in the hydride transfer in Escherichia coli dihydrofolate reductase
A key question concerning the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) is whether the Met20 loop is dynamically coupled to the chemical step during catalysis. A more basic, yet unanswered question is whether the Met20 loop adopts a closed conformation during the chemical hydride transfer step. To examine the most likely conformation of the Met20 loop during the chemical step, we studied the hydride transfer in wild type (WT) ecDHFR using hybrid quantum mechanics-molecular mechanics free energy simulations with the Met20 loop in a closed and disordered conformation.Single-molecule Förster resonance energy transfer (FRET) analysis discloses the dynamics of the DNA–topoisomerase II (Top2) interaction in the presence of TOP2-targeting agents
Topoisomerases play crucial roles in DNA replication, transcription, and recombination. For instance, topoisomerase II (Top2) is critically important for resolving DNA tangles during cell division, and as such, it is a broad anticancer drug target. Top2 regulates DNA topology by transiently breaking one double-stranded DNA molecule (cleavage), allowing a second double strand to pass through the opened DNA gate (opening), and then closing the gate by rejoining the broken ends. Drugs that modulate Top2 catalysis may therefore affect enzymatic activity at several different steps.Triphosphate Reorientation of the Incoming Nucleotide as a Fidelity Checkpoint in Viral RNA-dependent RNA Polymerases
The nucleotide incorporation fidelity of the viral RNA-dependent RNA polymerase (RdRp) is important for maintaining functional genetic information but, at the same time, is also important for generating sufficient genetic diversity to escape the bottlenecks of the host's antiviral response. We have previously shown that the structural dynamics of the motif D loop are closely related to nucleotide discrimination. Previous studies have also suggested that there is a reorientation of the triphosphate of the incoming nucleotide, which is essential before nucleophilic attack from the primer RNA 3′-hydroxyl.An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis
The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis.Structure of the Bacillus anthracis Sortase A Enzyme Bound to Its Sorting Signal: A FLEXIBLE AMINO-TERMINAL APPENDAGE MODULATES SUBSTRATE ACCESS
Background: The Bacillus anthracis sortase A (BaSrtA) enzyme attaches virulence factors to the cell surface.Results: The structure of BaSrtA bound to a substrate analog reveals key amino acids involved in substrate recognition and catalysis.Conclusion: BaSrtA modulates substrate access using a unique N-terminal appendage.Significance: This research could facilitate the design of new anti-infective agents that work by disrupting surface protein display.
