Type II restriction endonucleases: structure and mechanism

Type II restriction endonucleases are components of restriction modification systems that protect bacteria and archaea against invading foreign DNA. Most are homodimeric or tetrameric enzymes that cleave DNA at defined sites of 4–8 bp in length and require Mg²⁺ ions for catalysis. They differ in the details of the recognition process and the mode of cleavage, indicators that these enzymes are more diverse than originally thought. Still, most of them have a similar structural core and seem to share a common mechanism of DNA cleavage, suggesting that they evolved from a common ancestor. Only a few restriction endonucleases discovered thus far do not belong to the PD...D/ExK family of enzymes, but rather have active sites typical of other endonuclease families. The present review deals with new developments in the field of Type II restriction endonucleases. One of the more interesting aspects is the increasing awareness of the diversity of Type II restriction enzymes. Nevertheless, structural studies summarized herein deal with the more common subtypes. A major emphasis of this review will be on target site location and the mechanism of catalysis, two problems currently being addressed in the literature.Received 15 November 2004; accepted 9 December 2004     

Nucleotide binding domains of human CFTR: a structural classification of critical residues and disease-causing mutations.

Defective function of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes CF, the most frequent lethal inherited disease among the Caucasian population. The structure of this chloride ion channel includes two nucleotide-binding domains (NBDs), whose ATPase activity controls channel gating. Recently, the experimental structures of mouse and human CFTR NBD1 and our model of the human CFTR NBD1/NBD2 heterodimer have provided new insights into specific structural features of the CFTR NBD dimer. In the present work, we provide a structural classification of CF-causing mutations which may complement the existing functional classification. Our analysis also identified amino acid residues which may play a critical role in interdomain interaction and are located at the NBD1-NBD2 interface or on the surface of the dimer. In particular, a cluster of aromatic amino acids, which includes F508 and straddles the two NBDs, might be directly involved in the interaction of the NBD1/NBD2 heterodimer with the channel-forming membrane-spanning domains.Received 24 May 2005; received after revision 13 June 2005; accepted 18 June 2005     

From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance

The ATP binding cassette (ABC) superfamily of membrane transporters is one of the largest protein classes known, and counts numerous proteins involved in the trafficking of biological molecules across cell membranes. The first known human ABC transporter was P-glycoprotein (P-gp), which confers multidrug resistance (MDR) to anticancer drugs. In recent years, we have obtained an increased understanding of the mechanism of action of P-gp as its ATPase activity, substrate specificity and pharmacokinetic interactions have been investigated. This review focuses on the functional characterization of P-gp, as well as other ABC transporters involved in MDR: the family of multidrug-resistance-associated proteins (MRP1-7), and the recently discovered ABC half-transporter MXR (also known as BCRP, ABCP and ABCG2). We describe recent progress in the analysis of protein structure-function relationships, and consider the conceptual problem of defining and identifying substrates and inhibitors of MDR. An in-depth discussion follows of how coupling of nucleotide hydrolysis to substrate transport takes place, and we propose a scheme for the mechanism of P-gp function. Finally, the clinical correlations, both for reversal of MDR in cancer and for drug delivery, are discussed.     

Protein flexibility: its role in structure and mechanism revealed by molecular simulations

Computer simulations at the atomic level have arrived at a stage where they provide realistic modeling of flexibility in proteins (and the mobility of their associated solvent) that is important in understanding the nature of molecular motions. This can now be extended to the molecular and atomic motions that are associated with protein mechanisms. Moreover, the derived data agree reasonably accurately with experimental measurements of several kinetic and thermodynamic parameters. Fundamental insights emerge on the roles that this intrinsic flexibility plays in the thermodynamic characteristics of macromolecules in solution; these equip the investigator to probe the consequences of cognate interactions and ligand binding on entropy and enthalpy. Thus simulations can now provide a powerful tool for investigating protein mechanisms that complements the existing and the emerging experimental techniques. Received 29 May 2005; received after revision 23 August 2005; accepted 21 October 2005     

pHi regulatory ion transporters: an update on structure, regulation and cell function

Intracellular pH (pH_i) is a major regulator of various and critical cellular functions. A close regulation of pH_i is thus mandatory to maintain normal cellular activity. To this end, all cells express ion transporters that carry across their plasma membrane H⁺ or equivalent H⁺ into and out of the cell. Besides pH_i, these ion transporters are under the regulation of neurohormonal stimuli. This review summarises the molecular identity, regulation and function of the main membrane pH-regulatory ion transporters. Received 30 December 1998; received after revision 4 February 1999; accepted 9 February 1999     

The scorpine family of defensins: gene structure,alternative polyadenylation and fold recognition

Small cationic antimicrobial peptides (SCAMPs) as effectors of animal innate immunity provide the first defense against infectious pathogens. This class of molecules exists widely in invertebrate hemolymph and vertebrate skin secretion, but animal venoms are emerging as a new rich resource. Scorpine is a unique scorpion venom defensin peptide that has an extended amino-terminal sequence similar to cecropins. From the African scorpion Opistophthalmus carinatus venom gland, we isolated and identified several cDNAs encoding four new homologs of scorpine (named opiscorpines 1–4). Importantly, we show for the first time the existence of multiple opiscorpine mRNAs with variable 3 untranslated regions (UTRs) in the venom gland, which may be generated by alternative usage of polyadenylation signals. The complete opiscorpine gene structure including its promoter region is determined by genomic DNA amplification. Two large introns were found to be located within the 5 UTR and at the boundary of the mature peptide-coding region. Such a gene structure is distinct, when compared with other scorpion venom peptide genes. However, a comparative promoter analysis revealed that both opiscorpine and scorpion venom neurotoxins share a similar promoter organization. Sequence analysis and structural modeling allow us to group the scorpines and scorpion long-chain K-channel toxins together into one family that shares a similar fold with two distinct domains. The N-terminal cecropin-like domain displaying a clear antimicrobial activity implies that the scorpine family represents a group of real naturally occurring hybrids. Based on the phylogenetic analysis, a possible cooperative interaction between the N and C domains is elucidated, which provides an evolutionary basis for the design of a new class of anti-infectious drugs.Received 5 April 2004; accepted 17 May 2004     

N-acetylmuramic acid 6-phosphate lyases (MurNAc etherases): role in cell wall metabolism, distribution, structure, and mechanism

MurNAc etherases cleave the uniqued-lactyl ether bond of the bacterial cell wall sugar N-acetylmuramic acid (MurNAc). Members of this newly discovered family of enzymes are widely distributed among bacteria and are required to utilize peptidoglycan fragments obtained either from the environment or from the endogenous cell wall (i.e., recycling). MurNAc etherases are strictly dependent on the substrate MurNAc possessing a free reducing end and a phosphoryl group at C6. They carry a single conserved sugar phosphate isomerase/sugar phosphate- binding (SIS) domain to which MurNAc 6-phosphate is bound. Two subunits form an enzymatically active homodimer that structurally resembles the isomerase module of the double-SIS domain protein GlmS, the glucosamine 6-phosphate synthase. Structural comparison provides insights into the two-step lyase-type reaction mechanism of MurNAc etherases: β-elimination of the D-lactic acid substituent proceeds through a 2,3-unsaturated sugar intermediate to which water is subsequently added. Received 31 August 2007; received after revision 12 October 2007; accepted 1 November 2007