Protein Dynamics Enables Phosphorylation of Buried Residues in Cdk2/Cyclin-A-Bound p27

Proteins carry out a wide range of functions that are tightly regulated in space and time. Protein phosphorylation is the most common post-translation modification of proteins and plays a key role in the regulation of many biological processes. The finding that many phosphorylated residues are not solvent exposed in the unphosphorylated state opens several questions for understanding the mechanism that underlies phosphorylation and how phosphorylation may affect protein structures. First, because kinases need access to the phosphorylated residue, how do such buried residues become modified? Second, once phosphorylated, what are the structural effects of phosphorylation of buried residues, and do they lead to changed conformational dynamics? We have used the ternary complex between p27^Kip1 (p27), Cdk2, and cyclin A to study these questions using enhanced sampling molecular dynamics simulations. In line with previous NMR and single-molecule fluorescence experiments, we observe transient exposure of Tyr88 in p27, even in its unphosphorylated state. Once Tyr88 is phosphorylated, we observe a coupling to a second site, thus making Tyr74 more easily exposed and thereby the target for a second phosphorylation step. Our observations provide atomic details on how protein dynamics plays a role in modulating multisite phosphorylation in p27, thus supplementing previous experimental observations. More generally, we discuss how the observed phenomenon of transient exposure of buried residues may play a more general role in regulating protein function.     

Bone Formation and Resorption Are Both Increased in Experimental Autoimmune Arthritis

Introduction

Arthritic bone loss in the joints of patients with rheumatoid arthritis is the result of a combination of osteoclastic bone resorption and osteoblastic bone formation. This process is not completely understood, and especially the importance of local inflammation needs further investigation. We evaluated how bone formation and bone resorption are altered in experimental autoimmune arthritis.

Methods

Twenty-one female SKG mice were randomized to either an arthritis group or a control group. Tetracycline was used to identify mineralizing surfaces. After six weeks the right hind paws were embedded undecalcified in methylmethacrylate. The paws were cut exhaustively according to the principles of vertical sectioning and systematic sampling. 3D design-based methods were used to estimate the total number of osteoclasts, mineralizing surfaces, eroded surfaces, and osteoclast-covered bone surfaces. In addition the presence of adjacent inflammation was ascertained.

Results

The total number of osteoclasts, mineralizing surfaces, eroded surfaces, and osteoclast covered surfaces were elevated in arthritic paws compared to normal paws. Mineralizing surfaces were elevated adjacent to as well as not adjacent to inflammation in arthritic mice compared to normal mice. In arthritic mice, eroded surfaces and osteoclast covered surfaces were larger on bone surfaces adjacent to inflammation than on bone surfaces without adjacent inflammation. However, we found no difference between mineralizing surfaces at bone surfaces with or without inflammation in arthritic mice.

Conclusions

Inflammation induced an increase in resorptive bone surfaces as well as formative bone surfaces. The bone formative response may be more general, since formative bone surfaces were also increased when not associated with inflammation. Thus, the bone loss may be the result of a substantial local bone resorption, which cannot be compensated by the increased local bone formation. These findings may be valuable for the development of new osteoblast targeting drugs in RA.     

The relationships between Late Ordovician sea-level changes and faunal turnover in western Baltica: Geochemical evidence of oxic and dysoxic bottom-water conditions

The late Sandbian to the early Katian (Late Ordovician) in southeastern Norway is dominated by marine mudstones that contain an exceptional spectrum of macrofauna and trace element geochemistry, recording both abrupt and gradual faunal changes during major sea-level and environmental shifts. This study investigates the relationships between sea-level changes and their influence on the source of clastic material, oxygen levels in the bottom water and faunal changes, with special emphasis on the brachiopod fauna. Trace element ratios indicate that nearly stable upper dysoxic bottom-water conditions prevailed in the northwestern part of the Baltoscandian Sea. The exception is during the major shallowing in the earliest Katian, when there was an abrupt shift to oxic conditions as the sea bottom came within a normal storm wave base. Nonetheless, the pre-shallowing epibenthic fauna (though not the shelly endobenthic) in this area was rich and diverse and two major immigration phases of new brachiopod taxa are seen well before the shallowing. This indicates that the immigration of new taxa was not the result of an increase in oxygen content in the bottom water. More brachiopod genera stayed during the abrupt shallowing and increased oxic level than did during the following gradual return to deeper, dysoxic environments. The major brachiopod immigration phases took place markedly earlier in this northwestern part of the Baltoscandian Sea than in the central part (Eastern Baltic) and possibly also the comparable faunal turnover in Laurentia. The following disappearance of taxa during an early Katian transgressive event coincided with the faunal turnover in the shallow-water environments of the East Baltic.The depositional history was consistent within the investigated mudstone-dominated offshore facies of the Oslo Region, which are comparable to the Central Baltoscandian Confacies Belt of Baltoscandia. The composition of the siliciclastic material which was deposited in the basin was nearly constant through the sequence, though the land area expanded to include ophiolites, expressed by Cr enrichment, coinciding with the major sea-level drop. The Cr enrichment indicates that, by that time at least, the ophiolite complexes north of the Oslo Region became subaerially exposed. This enrichment thus forms a potential geochemical marker horizon representing the basal Katian Stage.     

Improved side‐chain torsion potentials for the Amber ff99SB protein force field

Recent advances in hardware and software have enabled increasingly long molecular dynamics (MD) simulations of biomolecules, exposing certain limitations in the accuracy of the force fields used for such simulations and spurring efforts to refine these force fields. Recent modifications to the Amber and CHARMM protein force fields, for example, have improved the backbone torsion potentials, remedying deficiencies in earlier versions. Here, we further advance simulation accuracy by improving the amino acid side‐chain torsion potentials of the Amber ff99SB force field. First, we used simulations of model alpha‐helical systems to identify the four residue types whose rotamer distribution differed the most from expectations based on Protein Data Bank statistics. Second, we optimized the side‐chain torsion potentials of these residues to match new, high‐level quantum‐mechanical calculations. Finally, we used microsecond‐timescale MD simulations in explicit solvent to validate the resulting force field against a large set of experimental NMR measurements that directly probe side‐chain conformations. The new force field, which we have termed Amber ff99SB‐ILDN, exhibits considerably better agreement with the NMR data. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Pressure profile and morphology of the arteries along the giraffe limb

Giraffes are the tallest animals on earth and the effects of gravity on their cardiovascular system have puzzled physiologists for centuries. The authors measured arterial and venous pressure in the foreleg of anesthetized giraffes, suspended in upright standing position, and determined the ratio between tunica media and lumen areas along the length of the femoral/tibial arteries in the hindleg. Volume fraction of elastin, density of vasa vasorum and innervations was estimated by stereology. Immunohistological staining with S100 was used to examine the innervation. The pressure increase in the artery and vein along the foreleg was not significantly different from what was expected on basis of gravity. The area of the arterial lumen in the hindleg decreased towards the hoof from 11.2 ± 4.2 to 0.6 ± 0.5 mm² (n = 10, P = 0.001), but most of this narrowing occurred within 2–4 cm immediately below the knee. This abrupt narrowing was associated with a marked increase in media to lumen area ratio (from 1.2 ± 0.5 to 7.8 ± 2.5; P = 0.001), and a decrease in mean volume fraction of elastin from 38 ± 6% proximal to the narrowing to 5.8 ± 1.1% distally (P = 0.001). The narrowing had a six-fold higher innervation density than the immediate distal and proximal regions. The sudden narrowing was also observed in the hind legs of neonates, indicating that it does not develop as an adaptation to the high transmural pressure in the standing giraffe. More likely it represents a preadaptation to the high pressures experienced by adult giraffes.