On the computation of the tertiary structure of globular proteins. III. Inter-residue distances and computed structures

An analysis of geometrical models for computing the tertiary structure of globular proteins from the primary structure is presented. The roles of initial configuration, input information on inter-residue distances and the errors in this information are delineated. It is shown that for local information like that on secondary structure, the calculated structure is very sensitive to errors and to the initial configuration. Thus, such information is far from adequate for predicting the tertiary structure. On the other hand, global information on all the inter-residue distances is quite insensitive to errors. A semi-empirical method is presented to estimate these distances and the calculated structures are given for two proteins—pancreatic trypsin inhibitor and parvalbumin. These structures have good resemblances to those determined by X-ray diffraction. A strategy for further refinement of the method is indicated.     

Extra EF Hand Unit (DX) Mediated Stabilization and Calcium Independency of α-Amylase

It is the common feature of α-amylases that calcium ion is required for their structural integrity and thermal stability. All amylases have at least one Ca²⁺ per molecule; therefore amino acids involved in calcium binding are specific and conserved. In this study, sequence analysis revealed the presence of EF-hand-like motif in calcium-binding loop of Bacillus megaterium WHO (BMW)-amylase that was previously isolated from BMW. The EF-hand motif and its variants (EF-hand-like motif) are the most common calcium-binding motifs found in a large number of protein families. To investigate the effect of calcium ion on the thermal stability and activity of BMW-amylase, we used site-directed mutagenesis to replace histidine 58 with Asp (D), Ile (I), Tyr (Y), Phe (F), and Arg (R) at the seventh position of EF-hand-like motif. Upon the addition of an extra DX unit to the calcium-binding loop in H58D variant, thermal stability, catalytic activity, and chelating power of the enzyme improved due to higher affinity toward calcium. H58D variant demonstrated calcium independency compared to the wild type and other created mutants. Conformational changes in the presence and absence of Ca²⁺ were monitored using fluorescence technique.     

Crystallography and mutagenesis point to an essential role for the N-terminus of human mitochondrial ClpP

We have determined a 2.1 A crystal structure for human mitochondrial ClpP (hClpP), the proteolytic component of the ATP-dependent ClpXP protease. HClpP has a structure similar to that of the bacterial enzyme, with the proteolytic active sites sequestered within an aqueous chamber formed by face-to-face assembly of the two heptameric rings. The hydrophobic N-terminal peptides of the subunits are bound within the narrow (12 A) axial channel, positioned to interact with unfolded substrates translocated there by the associated ClpX chaperone. Mutation or deletion of these residues causes a drastic decrease in ClpX-mediated protein and peptide degradation. Residues 8-16 form a mobile loop that extends above the ring surface and is also required for activity. The 28 amino acid C-terminal domain, a unique feature of mammalian ClpP proteins, lies on the periphery of the ring, with its proximal portion forming a loop that extends out from the ring surface. Residues at the start of the C-terminal domain impinge on subunit interfaces within the ring and affect heptamer assembly and stability. We propose that the N-terminal peptide of ClpP is a structural component of the substrate translocation channel and may play an important functional role as well.     

Roles of calcium ions in the activation and activity of the transglutaminase 3 enzyme

The transglutaminase 3 enzyme is widely expressed in many tissues including epithelia. We have shown previously that it can bind three Ca2+ ions, which in site one is constitutively bound, while those in sites two and three are acquired during activation and are required for activity. In particular, binding at site three opens a channel through the enzyme and exposes two tryptophan residues near the active site that are thought to be important for enzyme reaction. In this study, we have solved the structures of three more forms of this enzyme by x-ray crystallography in the presence of Ca2+ and/or Mg2+, which provide new insights on the precise contribution of each Ca2+ ion to activation and activity. First, we found that Ca2+ ion in site one can be exchanged with difficulty, and it has a binding affinity of Kd = 0.3 microm (DeltaH = -6.70 +/- 0.52 kcal/mol), which suggests it is important for the stabilization of the enzyme. Site two can be occupied by some lanthanides but only Ca2+ of the Group 2 family of alkali earth metals, and its occupancy are required for activity. Site three can be occupied by some lanthanides, Ca2+,or Mg2+; however, when Mg2+ is present, the enzyme is inactive, and the channel is closed. Thus Ca2+ binding in both sites two and three cooperate in opening the channel. We speculate that manipulation of the channel opening could be controlled by intracellular cation levels. Together, these data have important implications for reaction mechanism of the enzyme: the opening of a channel perhaps controls access to and manipulation of substrates at the active site.     

Enhanced cAMP-induced nitric oxide-dependent coronary dilation during myocardial stunning in conscious pigs

The goal of the current study was to determine the effects of cAMP-mediated coronary reactivity in conscious pigs with stunned myocardium induced by 1.5 h coronary stenosis (CS) and 12 h coronary artery reperfusion (CAR). Domestic swine (n = 5) were chronically instrumented with a coronary artery blood flow (CBF) probe, hydraulic occluder, left ventricular pressure gauge, wall-thickening crystals in the ischemic and nonischemic zones, and a coronary sinus catheter. The hydraulic occluder was inflated to induce a CS with a stable 38 +/- 1% reduction in CBF for 1.5 h. Before flow reduction and during CAR, cAMP-induced coronary vasodilation was investigated by forskolin (20 nmol. kg(-1). min(-1)). Enhanced CBF responses [+62 +/- 9%, P < 0.05, compared with pre-CS (+37 +/- 3%)] were observed for forskolin at 12 h after CAR as well as for bradykinin and reactive hyperemia. With the use of a similar protocol during systemic nitric oxide (NO) synthase inhibition with N(omega)-nitro-L-arginine (30 mg. kg(-1). day(-1) for 3 days), the enhanced CBF responses to forskolin, bradykinin, and reactive hyperemia were not observed after CS. Isolated microvessel preparations from pigs (n = 8) also demonstrated enhanced NO production to direct stimulation of adenylyl cyclase with forskolin (+71 +/- 12%) or NKH-477 (+60 +/- 10%) and administration of 8-bromo-cAMP (+74 +/- 13%), which were abolished by protein kinase A or NO synthase inhibition. These data indicate that cAMP stimulation elicits direct coronary vasodilation and that this action is amplified in the presence of sustained myocardial stunning after recovery from CS. This enhanced cAMP coronary vasodilation is mediated by an NO mechanism that may be involved in myocardial protection from ischemic injury.     

Effects of cardiogenic edema fluid on ion and fluid transport in the adult lung

We have previously shown that cardiogenic pulmonary edema fluid (EF) increases Na(+) and fluid transport by fetal distal lung epithelia (FDLE) (Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM and O'Brodovich H. J Physiol 544: 537-548, 2002). We now report the effect of EF on Na(+) and fluid transport by the adult lung. We first studied primary cultures of adult type II (ATII) epithelium and found that overnight exposure to EF increased Na(+) transport, and this effect was mainly due to factors other than catecholamines. Plasma did not stimulate Na(+) transport in ATII. Purification of EF demonstrated that at least some agent(s) responsible for the amiloride-insensitive component resided within the globulin fraction. ATII exposed to globulins demonstrated a conversion of amiloride-sensitive short-circuit current (I(sc)) to amiloride-insensitive I(sc) with no increase in total I(sc). Patch-clamp studies showed that ATII exposed to EF for 18 h had increased the number of highly selective Na(+) channels in their apical membrane. In situ acute exposure to EF increased the open probability of Na(+)-permeant ion channels in ATII within rat lung slices. EF did increase, by amiloride-sensitive pathways, the alveolar fluid clearance from the lungs of adult rats. We conclude that cardiogenic EF increases Na(+) transport by adult lung epithelia in primary cell culture, in situ and in vivo.     

Cxcl12 evolution--subfunctionalization of a ligand through altered interaction with the chemokine receptor

The active migration of primordial germ cells (PGCs) from their site of specification towards their target is a valuable model for investigating directed cell migration within the complex environment of the developing embryo. In several vertebrates, PGC migration is guided by Cxcl12, a member of the chemokine superfamily. Interestingly, two distinct Cxcl12 paralogs are expressed in zebrafish embryos and contribute to the chemotattractive landscape. Although this offers versatility in the use of chemokine signals, it also requires a mechanism through which migrating cells prioritize the relevant cues that they encounter. Here, we show that PGCs respond preferentially to one of the paralogs and define the molecular basis for this biased behavior. We find that a single amino acid exchange switches the relative affinity of the Cxcl12 ligands for one of the duplicated Cxcr4 receptors, thereby determining the functional specialization of each chemokine that elicits a distinct function in a distinct process. This scenario represents an example of protein subfunctionalization--the specialization of two gene copies to perform complementary functions following gene duplication--which in this case is based on receptor-ligand interaction. Such specialization increases the complexity and flexibility of chemokine signaling in controlling concurrent developmental processes.