The crystal structure of human procathepsin K

Cathepsin K is a cysteine protease present in human osteoclasts that plays an important role in bone resorption. Cathepsin K is synthesized as an inactive proenzyme and activated under conditions of low pH. Autoproteolytic processing of the N-terminal 99 amino acid propeptide produces the active, mature form of cathepsin K. It is presumed that the activation of procathepsin K in vivo occurs in the bone resorption pit, which has a low-pH environment. We have determined the structure of human procathepsin K at 2.8 A resolution. The structure of the mature enzyme domain within procathepsin K is virtually identical to that of mature cathepsin K. The fold of the propeptide of procathepsin K is similar to that observed in procathepsins B and L despite differences in length and sequence. A portion of the propeptide occupies the active site cleft of cathepsin K. Hydrophobic interactions, salt bridges, and hydrogen-bonding interactions are observed in the structure of the propeptide and between the propeptide and the mature enzyme of procathepsin K. These interactions suggest an explanation for the stability of the proenzyme. The structure of procathepsin K contributes to an understanding of the molecular basis of inhibition by the propeptide portion of the molecule and activation of this important member of the cysteine protease family.     

Simulations of reversible protein aggregate and crystal structure

We simulated the structure of reversible protein aggregates as a function of protein surface characteristics, protein-protein interaction energies, and the entropic penalty accompanying the immobilization of protein in a solid phase. These simulations represent an extension of our previous work on kinetically irreversible protein aggregate structure and are based on an explicit accounting of the specific protein-protein interactions that occur within reversible aggregates and crystals. We considered protein monomers with a mixture of hydrophobic and hydrophilic surface regions suspended in a polar solvent; the energetic driving force for aggregation is provided by the burial of solvent-exposed hydrophobic surface area. We analyzed the physical properties of the generated aggregates, including density, protein-protein contact distributions, solvent accessible surface area, porosity, and order, and compared our results with the protein crystallization literature as well as with the kinetically irreversible case. The physical properties of reversible aggregates were consonant with those observed for the irreversible aggregates, although in general, reversible aggregates were more stable energetically and were more crystal-like in their order content than their irreversible counterparts. The reversible aggregates were less dense than the irreversible aggregates, indicating that the increased energetic stability is derived primarily from the optimality rather than the density of the packing in the solid phase. The extent of hydrophobic protein-protein contacts and solvent-exposed surface area within the aggregate phase depended on the aggregation pathway: reversible aggregates tended to have a greater proportion of hydrophobic-hydrophobic contacts and a smaller fraction of hydrophobic solvent-exposed surface area. Furthermore, the arrangement of hydrophobic patches on the protein surface played a major role in the distribution of protein contacts and solvent content. This was readily reflected in the order of the aggregates: the greater the contiguity of the hydrophobic patches on the monomer surface, the less ordered the aggregates became, despite the opportunities for rearrangement offered by a reversible pathway. These simulations have enhanced our understanding of the impact of protein structural motifs on aggregate properties and on the demarcation between aggregation and crystallization.     

The high resolution crystal structure of deoxyhemoglobin S

We have refined the crystal structure of deoxyhemoglobin S (beta Glu6-->Val) at 2.05 A resolution to an R-factor of 16.5% (free R=21. 5%) using crystals isomorphous to those originally grown by Wishner and Love. A predominant feature of this crystal form is a double strand of hemoglobin tetramers that has been shown by a variety of techniques to be the fundamental building block of the intracellular sickle cell fiber. The double strand is stabilized by lateral contacts involving the mutant valine interacting with a pocket between the E and F helices on another tetramer. The new structure reveals some marked differences from the previously refined 3.0 A resolution structure, including several residues in the lateral contact which have shifted by as much as 3.5 A. The lateral contact includes, in addition to the hydrophobic interactions involving the mutant valine, hydrophilic interactions and bridging water molecules at the periphery of the contact. This structure provides further insights into hemoglobin polymerization and may be useful for the structure-based design of therapeutic agents to treat sickle cell disease.     

The crystal structure of bluetongue virus VP7

Bluetongue virus (BTV), a representative of the orbivirus genus of the Reoviridae, is considerably larger (at 80 nm across), and structurally more complex, than any virus for which we have comprehensive structural information. Orbiviruses infect mammalian hosts through insect vectors and cause economically important diseases of domesticated animals. They possess a segmented double-stranded RNA genome within a capsid composed of four major types of polypeptide chains. An outer layer of VP2 and VP5 is removed as the virus enters the target cell, to leave an intact core within the cell. This core is 70 nm across and composed of 780 copies of VP7 (M(r) 38K) that, as trimers, form 260 'bristly' capsomeres clothing an inner scaffold constructed from VP3 (M(r) 103K). We report here the crystal structure of VP7 from BTV serotype 10, which reveals a molecular architecture not seen previously in viral structural proteins. Each subunit consists of two domains, one a beta-sandwich, the other a bundle of alpha-helices, and a short carboxy-terminal arm which might tie trimers together during capsid formation. A concentration of methionine residues at the core of the molecule could provide plasticity, relieving structural mismatches during assembly.     

Projection structure of frog rhodopsin in two crystal forms

Rhodopsin is the G protein-coupled receptor that upon light activation triggers the visual transduction cascade. Rod cell outer segment disc membranes were isolated from dark-adapted frog retinas and were extracted with Tween detergents to obtain two-dimensional rhodopsin crystals for electron crystallography. When Tween 80 was used, tubular structures with a p2 lattice (a = 32 A, b = 83 A, gamma = 91 degrees) were formed. The use of a Tween 80/Tween 20 mixture favored the formation of larger p22(1)2(1) lattices (a = 40 A, b = 146 A, gamma = 90 degrees). Micrographs from frozen hydrated frog rhodopsin crystals were processed, and projection structures to 7-A resolution for the p22(1)2(1) form and to 6-A resolution for the p2 form were calculated. The maps of frog rhodopsin in both crystal forms are very similar to the 9-A map obtained previously for bovine rhodopsin and show that the arrangement of the helices is the same. In a tentative topographic model, helices 4, 6, and 7 are nearly perpendicular to the plane of the membrane. In the higher-resolution projection maps of frog rhodopsin, helix 5 looks more tilted than it appeared previously. The quality of the two frog rhodopsin crystals suggests that they would be suitable to obtain a three-dimensional structure in which all helices would be resolved.     

Refined crystal structure and mutagenesis of human granulocyte-macrophage colony-stimulating factor

We reviewed the clinical and technical outcomes of 25 patients with neuromuscular scoliosis, who were treated by Luque instrumentation and posterior spinal fusion from the upper thoracic spine to L5 between 1981 and 1988. A mean curve correction of 46% was obtained operatively with a mean 8 degrees loss of correction during the follow-up period that ranged from 1.9 to 9.4 years (mean, 5.5). Pelvic obliquity was improved 50% from a mean of 16.1 degrees to a mean of 8.1 degrees in 24 patients for whom data were available. At final follow-up, the mean pelvic obliquity increased to 11.4 degrees with only two patients increasing > 8 degrees. The cause for major postoperative increase in pelvic obliquity was continued anterior spinal growth with torsion of the fusion mass and was not related to changes limited to the L5-S1 motion segment. Posterior fusion and instrumentation from the upper thoracic spine to L5 without anterior fusion provides adequate correction and control of spinal deformity for many patients with cerebral palsy. Those patients with significant growth remaining, or with severe deformities, may benefit by preliminary anterior release and fusion or inclusion of the pelvis and sacrum.     

Fibrillar crystal structure in essential monoclonal IgM kappa cryoglobulinemia

OBJECTIVE: To study erysipelas in farmed emus and the treatment and control of the disease by vaccination. DESIGN: A retrospective study of field outbreaks in emus and challenge experiments in mice using field and vaccine strains of the organism. PROCEDURE: Outbreaks of the disease were described. Field strains of the organism were identified and tested by challenge experiments in mice against commercial vaccine strains. RESULTS: Erysipelas was characterised by sudden death in yearling emus. Deaths mainly occurred during the cold wet months. Mortalities of 6 to 10% were seen within the first 7 to 10 days of an outbreak. Clinical signs were uncommon but some birds exhibited lethargy and greenish diarrhoea. Necropsy findings included marked petechial haemorrhages on the serosal surface of the large intestine in particular, pericardial effusion and congestion and mottling of the liver. Treatment consisted of individual or mass medication with procaine penicillin, reduction of stress factors such as overcrowding, and spelling and rotation of paddocks. Isolates from two field outbreaks were identified as strain 21. Complete protection was provided by a commercial strain 2b vaccine against challenge by strain 21 field isolates in mice. Annual vaccination of birds at 4 weeks and again at 8 weeks of age appeared to control further outbreaks on farms where the disease had previously occurred and vaccination appeared to protect for at least 12 months.     

X-ray crystal structure of glycinamide ribonucleotide synthetase from Escherichia coli

Glycinamide ribonucleotide synthetase (GAR-syn) catalyzes the second step of the de novo purine biosynthetic pathway; the conversion of phosphoribosylamine, glycine, and ATP to glycinamide ribonucleotide (GAR), ADP, and Pi. GAR-syn containing an N-terminal polyhistidine tag was expressed as the SeMet incorporated protein for crystallographic studies. In addition, the protein as isolated contains a Pro294Leu mutation. This protein was crystallized, and the structure solved using multiple-wavelength anomalous diffraction (MAD) phase determination and refined to 1.6 A resolution. GAR-syn adopts an alpha/beta structure that consists of four domains labeled N, A, B, and C. The N, A, and C domains are clustered to form a large central core structure whereas the smaller B domain is extended outward. Two hinge regions, which might readily facilitate interdomain movement, connect the B domain and the main core. A search of structural databases showed that the structure of GAR-syn is similar to D-alanine:D-alanine ligase, biotin carboxylase, and glutathione synthetase, despite low sequence similarity. These four enzymes all utilize similar ATP-dependent catalytic mechanisms even though they catalyze different chemical reactions. Another ATP-binding enzyme with low sequence similarity but unknown function, synapsin Ia, was also found to share high structural similarity with GAR-syn. Interestingly, the GAR-syn N domain shows similarity to the N-terminal region of glycinamide ribonucleotide transformylase and several dinucleotide-dependent dehydrogenases. Models of ADP and GAR binding were generated based on structure and sequence homology. On the basis of these models, the active site lies in a cleft between the large domain and the extended B domain. Most of the residues that facilitate ATP binding belong to the A or B domains. The N and C domains appear to be largely responsible for substrate specificity. The structure of GAR-syn allows modeling studies of possible channeling complexes with PPRP amidotransferase.