Meindert H. Lamers

The Crystal Structure of DNA Mismatch Repair Protein Muts Binding to a G X T Mismatch

DNA mismatch repair ensures genomic integrity on DNA replication. Recognition of a DNA mismatch by a dimeric MutS protein initiates a cascade of reactions and results in repair of the newly synthesized strand; however, details of the molecular mechanism remain controversial. Here we present the crystal structure at 2.2 Å of MutS from Escherichia coli bound to a G·T mismatch. The two MutS monomers have different conformations and form a heterodimer at the structural level. Only one monomer recognizes the mismatch specifically and has ADP bound. Mismatch recognition occurs by extensive minor groove interactions causing unusual base pairing and kinking of the DNA. Nonspecific major groove DNA-binding domains from both monomers embrace the DNA in a clamp-like structure. The interleaved nucleotide-binding sites are located far from the DNA. Mutations in human MutSα (MSH2/MSH6) that lead to hereditary predisposition for cancer, such as hereditary non-polyposis colorectal cancer, can be mapped to this crystal structure.

Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment.

Signaling by the epidermal growth factor receptor requires an allosteric interaction between the kinase domains of two receptors, whereby one activates the other. We show that the intracellular juxtamembrane segment of the receptor, known to potentiate kinase activity, is able to dimerize the kinase domains. The C-terminal half of the juxtamembrane segment latches the activated kinase domain to the activator, and the N-terminal half of this segment further potentiates dimerization, most likely by forming an antiparallel helical dimer that engages the transmembrane helices of the activated receptor. Our data are consistent with a mechanism in which the extracellular domains block the intrinsic ability of the transmembrane and cytoplasmic domains to dimerize and activate, with ligand binding releasing this block. The formation of the activating juxtamembrane latch is prevented by the C-terminal tails in a structure of an inactive kinase domain dimer, suggesting how alternative dimers can prevent ligand-independent activation.

Crystal Structure of the Catalytic α Subunit of E. coli Replicative DNA Polymerase III

Bacterial replicative DNA polymerases such as Polymerase III (Pol III) share no sequence similarity with other polymerases. The crystal structure, determined at 2.3 A resolution, of a large fragment of Pol III (residues 1-917), reveals a unique chain fold with localized similarity in the catalytic domain to DNA polymerase beta and related nucleotidyltransferases. The structure of Pol III is strikingly different from those of members of the canonical DNA polymerase families, which include eukaryotic replicative polymerases, suggesting that the DNA replication machinery in bacteria arose independently. A structural element near the active site in Pol III that is not present in nucleotidyltransferases but which resembles an element at the active sites of some canonical DNA polymerases suggests that, at a more distant level, all DNA polymerases may share a common ancestor. The structure also suggests a model for interaction of Pol III with the sliding clamp and DNA.

The alternating ATPase domains of MutS control DNA mismatch repair

DNA mismatch repair is an essential safeguard of genomic integrity by removing base mispairings that may arise from DNA polymerase errors or from homologous recombination between DNA strands. In Escherichia coli, the MutS enzyme recognizes mismatches and initiates repair. MutS has an intrinsic ATPase activity crucial for its function, but which is poorly understood. We show here that within the MutS homodimer, the two chemically identical ATPase sites have different affinities for ADP, and the two sites alternate in ATP hydrolysis. A single residue, Arg697, located at the interface of the two ATPase domains, controls the asymmetry. When mutated, the asymmetry is lost and mismatch repair in vivo is impaired. We propose that asymmetry of the ATPase domains is an essential feature of mismatch repair that controls the timing of the different steps in the repair cascade.

The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase

BackgroundSoluble guanylate cyclases generate cyclic GMP when bound to nitric oxide, thereby linking nitric oxide levels to the control of processes such as vascular homeostasis and neurotransmission. The guanylate cyclase catalytic module, for which no structure has been determined at present, is a class III nucleotide cyclase domain that is also found in mammalian membrane-bound guanylate and adenylate cyclases.ResultsWe have determined the crystal structure of the catalytic domain of a soluble guanylate cyclase from the green algae Chlamydomonas reinhardtii at 2.55 Å resolution, and show that it is a dimeric molecule.ConclusionComparison of the structure of the guanylate cyclase domain with the known structures of adenylate cyclases confirms the close similarity in architecture between these two enzymes, as expected from their sequence similarity. The comparison also suggests that the crystallized guanylate cyclase is in an inactive conformation, and the structure provides indications as to how activation might occur. We demonstrate that the two active sites in the dimer exhibit positive cooperativity, with a Hill coefficient of ~1.5. Positive cooperativity has also been observed in the homodimeric mammalian membrane-bound guanylate cyclases. The structure described here provides a reliable model for functional analysis of mammalian guanylate cyclases, which are closely related in sequence.

ATP increases the affinity between MutS ATPase domains: Implications for ATP hydrolysis and conformational changes

MutS is the key protein of the Escherichia coli DNA mismatch repair system. It recognizes mispaired and unpaired bases and has intrinsic ATPase activity. ATP binding after mismatch recognition by MutS serves as a switch that enables MutL binding and the subsequent initiation of mismatch repair. However, the mechanism of this switch is poorly understood. We have investigated the effects of ATP binding on the MutS structure. Crystallographic studies of ATP-soaked crystals of MutS show a trapped intermediate, with ATP in the nucleotide-binding site. Local rearrangements of several residues around the nucleotide-binding site suggest a movement of the two ATPase domains of the MutS dimer toward each other. Analytical ultracentrifugation experiments confirm such a rearrangement, showing increased affinity between the ATPase domains upon ATP binding and decreased affinity in the presence of ADP. Mutations of specific residues in the nucleotide-binding domain reduce the dimer affinity of the ATPase domains. In addition, ATP-induced release of DNA is strongly reduced in these mutants, suggesting that the two activities are coupled. Hence, it seems plausible that modulation of the affinity between ATPase domains is the driving force for conformational changes in the MutS dimer. These changes are driven by distinct amino acids in the nucleotide-binding site and form the basis for long-range interactions between the ATPase domains and DNA-binding domains and subsequent binding of MutL and initiation of mismatch repair.

Structure and regulatory mechanism of Aquifex aeolicus NtrC4: variability and evolution in bacterial transcriptional regulation.

Genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. Comparative studies provide insights into the functional consequences of specific changes. Here we present structural and biochemical studies of NtrC4, a sigma-54 activator from Aquifex aeolicus, and compare it with NtrC1 (a paralog) and NtrC (a homolog from Salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may have occurred. Activity assays show that assembly of NtrC4s active oligomer is repressed by the N-terminal receiver domain, and that BeF3- addition (mimicking phosphorylation) removes this repression. Observation of assembly without activation for NtrC4 indicates that it is much less strongly repressed than NtrC1. The crystal structure of the unactivated receiver-ATPase domain combination shows a partially disrupted interface. NMR structures of the regulatory domain show that its activation mechanism is very similar to that of NtrC1. The crystal structure of the NtrC4 DNA-binding domain shows that it is dimeric and more similar in structure to NtrC than NtrC1. Electron microscope images of the ATPase-DNA-binding domain combination show formation of oligomeric rings. Sequence alignments provide insights into the distribution of activation mechanisms in this family of proteins.

Architecture of the Pol III–clamp–exonuclease complex reveals key roles of the exonuclease subunit in processive DNA synthesis and repair

DNA polymerase III (Pol III) is the catalytic α subunit of the bacterial DNA Polymerase III holoenzyme. To reach maximum activity, Pol III binds to the DNA sliding clamp β and the exonuclease ε that provide processivity and proofreading, respectively. Here, we characterize the architecture of the Pol III–clamp–exonuclease complex by chemical crosslinking combined with mass spectrometry and biochemical methods, providing the first structural view of the trimeric complex. Our analysis reveals that the exonuclease is sandwiched between the polymerase and clamp and enhances the binding between the two proteins by providing a second, indirect, interaction between the polymerase and clamp. In addition, we show that the exonuclease binds the clamp via the canonical binding pocket and thus prevents binding of the translesion DNA polymerase IV to the clamp, providing a novel insight into the mechanism by which the replication machinery can switch between replication, proofreading, and translesion synthesis.

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader

The DNA replication machinery is an important target for antibiotic development in increasingly drug-resistant bacteria, including Mycobacterium tuberculosis. Although blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In Escherichia coli, the proofreading subunit of the replisome, the ɛ exonuclease, is essential for high-fidelity DNA replication; however, we find that the corresponding subunit is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase DnaE1 itself encodes an editing function that proofreads DNA replication, mediated by an intrinsic 3′–5′ exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by more than 3,000-fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP domain–mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader.

A structural role for the PHP domain in E. coli DNA polymerase III

BackgroundIn addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive.ResultsGenomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 Å resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity.ConclusionsWhile the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase.