Preparation, characterization, and the crystal structure of the inhibitor ZK-807834 (CI-1031) complexed with factor Xa

Factor Xa plays a critical role in the formation of blood clots. This serine protease catalyzes the conversion of prothrombin to thrombin, the first joint step that links the intrinsic and extrinsic coagulation pathways. There is considerable interest in the development of factor Xa inhibitors for the intervention in thrombic diseases. This paper presents the structure of the inhibitor ZK-807834, also known as CI-1031, bound to factor Xa and provides the details of the protein purification and crystallization. Results from mass spectrometry indicate that the factor Xa underwent autolysis during crystallization and the first EGF-like domain was cleaved from the protein. The crystal structure of the complex shows that the amidine of ZK-807834 forms a salt bridge with Asp189 in the S1 pocket and the basic imidazoline fits snugly into the S4 site. The central pyridine ring provides a fairly rigid linker between these groups. This rigidity helps minimize entropic losses during binding. In addition, the structure reveals new interactions that were not found in the previous factor Xa/inhibitor complexes. ZK-807834 forms a strong hydrogen bond between an ionized 2-hydroxy group and Ser195 of factor Xa. There is also an aromatic ring-stacking interaction between the inhibitor and Trp215 in the S4 pocket. These interactions contribute to both the potency of this compound (K(I) = 0.11 nM) and the >2500-fold selectivity against homologous serine proteases such as trypsin.     

Partitioning and diversity of nuclear and organelle markers in native and introduced populations of Epipactis helleborine (Orchidaceae)

Variability of allozymes (1170 individuals, 47 populations) and chloroplast DNA (692 individuals, 29 populations) was examined in native European and introduced North American populations of Epipactis helleborine (Orchidaceae). At the species level, the percentage of allozyme loci that were polymorphic (P(99)) was 67%, with a mean of 2.11 alleles (A) per locus, and an expected heterozygosity (H(exp)) of 0.294. At the population level, mean P(99) = 56%, mean A = 1.81, and mean H(exp) = 0.231. Although field observations suggest that self-pollination occurs frequently, populations had a genetic structure consistent with Hardy-Weinberg expectations and random mating (mean F(IS) = 0.002). There was significant deviation from panmixia associated with population differentiation (mean F(ST) = 0.206). The distribution of two chloroplast haplotypes showed that 15 of the 29 populations were polymorphic. Using both nuclear and organelle F(ST) estimates, a pollen to seed flow ratio of 1.43?:?1 was calculated. This is very low compared with published estimates for other plant groups, consistent with the high dispersability of orchid seeds. Finally, there was no evidence for a genetic bottleneck associated with the introduction of E. helleborine to North America.     

Electrophysiological characterization and ionic stoichiometry of the rat brain K(+)-dependent NA(+)/CA(2+) exchanger, NCKX2

We have recently described a novel K(+)-dependent Na(+)/Ca(2+) exchanger, NCKX2, that is abundantly expressed in brain neurons (Tsoi, M., Rhee, K.-H., Bungard, D., Li, X.-F., Lee, S.-L., Auer, R. N., and Lytton, J. (1998) J. Biol. Chem. 273, 4115--4162). The precise role for NCKX2 in neuronal Ca(2+) homeostasis is not yet clearly understood but will depend upon the functional properties of the molecule. Here, we have performed whole-cell patch clamp analysis to characterize cation dependences and ion stoichiometry for rat brain NCKX2, heterologously expressed in HEK293 cells. Outward currents generated by reverse NCKX2 exchange depended on external Ca(2+) with a K(12) of 1.4 or 101 microm without or with 1 mm Mg(2+), and on external K(+) with a K(1/2) of about 12 or 36 mm with choline or Li(+) as counter ion, respectively. Na(+) inhibited outward currents with a K(1/2) of about 60 mm. Inward currents generated by forward NCKX2 exchange depended upon external Na(+) with a K(1/2) of 30 mm and a Hill coefficient of 2.8. K(+) inhibited the inward currents by a maximum of 40%, with a K(1/2) of 2 mm or less, depending upon the conditions. The transport stoichiometry of NCKX2 was determined by observing the change in reversal potential as individual ion gradients were altered. Our data support a stoichiometry for rat brain NCKX2 of 4 Na(+):(1 Ca(2+) + 1 K(+)). These findings provide the first electrophysiological characterization of rat brain NCKX2, and the first evidence that a single recombinantly expressed NCKX polypeptide encodes a K(+)-transporting Na(+)/Ca(2+) exchanger with a transport stoichiometry of 4 Na(+):(1 Ca(2+) + 1 K(+)).     

Manipulating the genetic code for membrane protein production: What have we learnt so far?

With synthetic gene services, molecular cloning is as easy as ordering a pizza. However choosing the right RNA code for efficient protein production is less straightforward, more akin to deciding on the pizza toppings. The possibility to choose synonymous codons in the gene sequence has ignited a discussion that dates back 50years: Does synonymous codon use matter? Recent studies indicate that replacement of particular codons for synonymous codons can improve expression in homologous or heterologous hosts, however it is not always successful. Furthermore it is increasingly apparent that membrane protein biogenesis can be codon-sensitive. Single synonymous codon substitutions can influence mRNA stability, mRNA structure, translational initiation, translational elongation and even protein folding. Synonymous codon substitutions therefore need to be carefully evaluated when membrane proteins are engineered for higher production levels and further studies are needed to fully understand how to select the codons that are optimal for higher production. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

The evolution of filamin - A protein domain repeat perspective

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates.     

Parallel Markov chain Monte Carlo - bridging the gap to high-performance Bayesian computation in animal breeding and genetics

Background

Most Bayesian models for the analysis of complex traits are not analytically tractable and inferences are based on computationally intensive techniques. This is true of Bayesian models for genome-enabled selection, which uses whole-genome molecular data to predict the genetic merit of candidate animals for breeding purposes. In this regard, parallel computing can overcome the bottlenecks that can arise from series computing. Hence, a major goal of the present study is to bridge the gap to high-performance Bayesian computation in the context of animal breeding and genetics.

Results

Parallel Monte Carlo Markov chain algorithms and strategies are described in the context of animal breeding and genetics. Parallel Monte Carlo algorithms are introduced as a starting point including their applications to computing single-parameter and certain multiple-parameter models. Then, two basic approaches for parallel Markov chain Monte Carlo are described: one aims at parallelization within a single chain; the other is based on running multiple chains, yet some variants are discussed as well. Features and strategies of the parallel Markov chain Monte Carlo are illustrated using real data, including a large beef cattle dataset with 50K SNP genotypes.

Conclusions

Parallel Markov chain Monte Carlo algorithms are useful for computing complex Bayesian models, which does not only lead to a dramatic speedup in computing but can also be used to optimize model parameters in complex Bayesian models. Hence, we anticipate that use of parallel Markov chain Monte Carlo will have a profound impact on revolutionizing the computational tools for genomic selection programs.     

Temporal and spatial dynamics of competitive parapatry in chewing lice

We synthesize observations from 1979 to 2016 of a contact zone involving two subspecies of pocket gophers (Thomomys bottae connectens and T. b. opulentus) and their respective chewing lice (Geomydoecus aurei and G. centralis) along the Río Grande Valley in New Mexico, U.S.A., to test predictions about the dynamics of the zone. Historically, the natural flood cycle of the Rio Grande prevented contact between the two subspecies of pocket gophers. Flood control measures completed in the 1930s permitted contact, thus establishing the hybrid zone between the pocket gophers and the contact zone between their lice (without hybridization). Since that time, the pocket gopher hybrid zone has stabilized, whereas the northern chewing louse species has replaced the southern louse species at a consistent rate of ~150 m/year. The 0.2–0.8 width of the replacement zone has remained constant, reflecting the constant rate of chewing louse species turnover on a single gopher and within a local pocket gopher population. In contrast, the full width of the replacement zone (northernmost G. centralis to southernmost G. aurei) has increased annually. By employing a variety of metrics of the species replacement zone, we are better able to understand the dynamics of interactions between and among the chewing lice and their pocket gopher hosts. This research provides an opportunity to observe active species replacement and resulting distributional shifts in a parasitic organism in its natural setting.