Pepsin-like aspartic proteases (PAPs) as model systems for combining biomolecular simulation with biophysical experiments

AbstractFlexibility of β hairpin structure known as flap plays a key role in catalytic activity and substrate intake in pepsin-like aspartic proteases. Most of these enzymes share structural and sequence similarity. Tyrosine is a conserved residue present in the flap region of pepsin-like aspartic proteases. In apo protease, tyrosine remains in a dynamic equilibrium between normal and flipped states due to rotation of χ1 angle (distributions of the χ1 angle centred around radian or radian are denoted as normal, whereas distribution centred around ±π radian is denoted as flipped). In this study, we have used apo Plm-II and BACE-1 as model systems. Independent MD simulations starting with Plm-II and BACE-1 remained stuck either in normal or flipped state. Metadynamics simulations using torsion angles (χ1 and χ2 of Tyr) as CVs sampled transition between normal and flipped states. Qualitatively, flipped and normal states predicted to be equally populated. These states were stabilised by H-bond interactions to tryptophan (normal) and catalytic aspartate (flipped) respectively. Further, mutation of tyrosine to an amino-acid with smaller side-chain, such as alanine; reduced flap flexibility and resulted in a flap collapse. This is in accordance with previous experimental studies which showed that mutation to alanine resulted in loss of activity in pepsin-like aspartic proteases. Using apo plasmepsin-II and BACE-1 as model systems, we have hypothesised that the rotation of tyrosine side-chain is the key movement which governs the flap dynamics in all pepsin-like aspartic proteases.

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Virus-Like Particles in a Human Breast Cancer: Structural Similarity to Previously Reported Particles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100072332 ◽

1973 ◽

Vol 31 ◽

pp. 378-379

Author(s):

G. Kasnic ◽

S. E. Stewart ◽

C. Urbanski

Keyword(s):

Breast Cancer ◽

Biological Activity ◽

Human Breast Cancer ◽

Osteogenic Sarcoma ◽

Human Tumor ◽

Structural Similarity ◽

Cell Tumor ◽

Human Breast ◽

Human Tumor Cell ◽

Type Particle

We have reported the maturation of an intracisternal A-type particle in murine plasma cell tumor cultures and three human tumor cell cultures (rhabdomyosarcoma, lung adenocarcinoma, and osteogenic sarcoma) after IUDR-DMSO activation. In all of these studies the A-type particle seems to develop into a form with an electron dense nucleoid, presumably mature, which is also intracisternal. A similar intracisternal A-type particle has been described in leukemic guinea pigs. Although no biological activity has yet been demonstrated for these particles, on morphologic grounds, and by the manner in which they develop within the cell, they may represent members of the same family of viruses.

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Structural similarity of ribosomes from E. coli and A. salina

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082078 ◽

1978 ◽

Vol 36 (2) ◽

pp. 242-243

Author(s):

M. Boublik ◽

R.M. Wydro ◽

W. Hellmann ◽

F. Jenkins

Keyword(s):

Ribosomal Proteins ◽

Direct Evidence ◽

Structural Similarity ◽

Carbon Support ◽

Artemia Salina ◽

Uranyl Acetate ◽

Biological Assays ◽

E Coli ◽

And Function ◽

Fine Carbon

Ribosomes are ribonucleoprotein particles necessary for processing the genetic information of mRNA into proteins. Analogy in composition and function of ribosomes from diverse species, established by biochemical and biological assays, implies their structural similarity. Direct evidence obtained by electron microscopy seems to be of increasing relevance in understanding the structure of ribosomes and the mechanism of their role in protein synthesis.The extent of the structural homology between prokaryotic and eukaryotic ribosomes has been studied on ribosomes of Escherichia coli (E.c.) and Artemia salina (A.s.). Despite the established differences in size and in the amount and proportion of ribosomal proteins and RNAs both types of ribosomes show an overall similarity. The monosomes (stained with 0.5% aqueous uranyl acetate and deposited on a fine carbon support) appear in the electron micrographs as round particles with a diameter of approximately 225Å for the 70S E.c. (Fig. 1) and 260Å for the 80S A.s. monosome (Fig. 2).

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Nuclear coiled bodies and the nucleolus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100167834 ◽

1994 ◽

Vol 52 ◽

pp. 20-21

Author(s):

K. Brasch ◽

J. Williams ◽

D. Gallo ◽

T. Lee ◽

R. L. Ochs

Keyword(s):

Mouse Liver ◽

Nuclear Body ◽

Nuclear Bodies ◽

Model Systems ◽

Coiled Body ◽

Rrna Processing ◽

Malignant Cells ◽

Sm Proteins ◽

Coiled Bodies ◽

Unique Protein

Though first described in 1903 by Ramon-y-Cajal as silver-staining “accessory bodies” to nucleoli, nuclear bodies were subsequently rediscovered by electron microscopy about 30 years ago. Nuclear bodies are ubiquitous, but seem most abundant in hyperactive and malignant cells. The best studied type of nuclear body is the coiled body (CB), so termed due to characteristic morphology and content of a unique protein, p80-coilin (Fig.1). While no specific functions have as yet been assigned to CBs, they contain spliceosome snRNAs and proteins, and also the nucleolar protein fibrillarin. In addition, there is mounting evidence that CBs arise from or are generated near the nucleolus and then migrate into the nucleoplasm. This suggests that as yet undefined links may exist, between nucleolar pre-rRNA processing events and the spliceosome-associated Sm proteins in CBs.We are examining CB and nucleolar changes in three diverse model systems: (1) estrogen stimulated chick liver, (2) normal and neoplastic cells, and (3) polyploid mouse liver.

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Microtubule Dynamics and Organelle Transport in Reticulomyxa

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158510 ◽

1990 ◽

Vol 48 (3) ◽

pp. 194-195

Author(s):

Yih-Tai Chen ◽

Ursula Euteneuer ◽

Ken B. Johnson ◽

Michael P. Koonce ◽

Manfred Schliwa

Keyword(s):

Plasma Membrane ◽

Light Microscopy ◽

Cytoplasmic Dynein ◽

Living Cells ◽

Microtubule Dynamics ◽

Model Systems ◽

Organelle Transport ◽

Biochemical Characteristics ◽

Dependent Transport

The application of video techniques to light microscopy and the development of motility assays in reactivated or reconstituted model systems rapidly advanced our understanding of the mechanism of organelle transport and microtubule dynamics in living cells. Two microtubule-based motors have been identified that are good candidates for motors that drive organelle transport: kinesin, a plus end-directed motor, and cytoplasmic dynein, which is minus end-directed. However, the evidence that they do in fact function as organelle motors is still indirect.We are studying microtubule-dependent transport and dynamics in the giant amoeba, Reticulomyxa. This cell extends filamentous strands backed by an extensive array of microtubules along which organelles move bidirectionally at up to 20 μm/sec (Fig. 1). Following removal of the plasma membrane with a mild detergent, organelle transport can be reactivated by the addition of ATP (1). The physiological, pharmacological and biochemical characteristics show the motor to be a cytoplasmic form of dynein (2).

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Microstructural observations of the “Wustite-Spinel” coexistence following quenching of cation-excess spinels, Ni2(1+x)Ti1-xO4

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100178422 ◽

1990 ◽

Vol 48 (4) ◽

pp. 1058-1059

Author(s):

Ian M. Anderson ◽

Arnulf Muan ◽

C. Barry Carter

Keyword(s):

Film Growth ◽

Spinel Phase ◽

Model Systems ◽

Ion Milling ◽

Coexistence Of Phases ◽

Nonequilibrium Conditions ◽

Diffraction Patterns ◽

Spinel Structures ◽

Highly Porous ◽

Standard Techniques

Oxide mixtures which feature a coexistence of phases with the wüstite and spinel structures are considered model systems for the study of solid-state reaction kinetics, phase boundaries, and thin-film growth, and such systems are especially suited to TEM studies. (In this paper, the terms “wüstite” and “spinel” will refer to phases of those structure types.) The study of wüstite-spinel coexistence has been limited mostly to systems near their equilibrium condition, where the assumptions of local thermodynamic equilibrium are valid. The cation-excess spinels of the type Ni2(1+x)Ti1-xO4, which reportedly exist only above 1375°C4, provide an excellent system for the study of wüstite-spinel coexistence under highly nonequilibrium conditions. The nature of these compounds has been debated in the literature. X-ray and neutron powder diffraction patterns have been used to advocate the existence of a single-phase, non- stoichiometric spinel. TEM studies of the microstructure have been used to suggest equilibrium coexistence of a stoichiometric spinel, Ni2TiO4, and a wüstite phase; this latter study has shown a coexistence of wüstite and spinel phases in specimens thought to have been composed of a single, non- stoichiometric spinel phase. The microstructure and nature of this phase coexistence is the focus of this study. Specimens were prepared by ball-milling a mixture of NiO and TiO2 powders with 10 wt.% TiO2. The mixture was fired in air at 1483°C for 5 days, and then quenched to room temperature. The aggregate thus produced was highly porous, and needed to be infiltrated prior to TEM sample preparation, which was performed using the standard techniques of lapping, dimpling, and ion milling.

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Role of the Small (40S) Subunits in the Structure of the Interface of Eukaryotic Ribosomes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002427 ◽

1980 ◽

Vol 38 ◽

pp. 548-549

Author(s):

M. Boublik ◽

N. Robakis ◽

W. Hellmann ◽

F. Jenkins

Keyword(s):

Structural Similarity ◽

High Resolution Electron Microscopy ◽

Peptide Bond ◽

Uranyl Acetate ◽

Mutual Orientation ◽

Mutual Position ◽

Peptide Bond Formation ◽

Resolution Electron ◽

Direct Technique ◽

Structural Details

Ribosomes are ribonucleoprotein particles which process the genetic information coded in mRNA into protein synthesis. The analogy in function and composition of ribosomes from various sources, both prokaryotic and eukaryo-tic, imply a structural similarity. At present, high resolution electron microscopy is the most direct technique with a potential to resolve the extent of the structural homology of ribosomal particles at a macromolecular level. The structure of ribosomes is highly complex as a result of the large number of their constituents. In general, 80S eukaryotic monosomes consist of two uneven subunits - large (60S) and small (40S) - accomodating four different RNAs and approximately 80 different proteins. Mutual orientation of both subunits on the monosome is of particular interest because it determines the interface, the supposed site of interactions of ribosomes with other macro-molecules involved in peptide bond formation. Since entrapping of the contrasting solution (0.5% aqueous uranyl acetate) obscures all structural details in the interface, information on its architecture is limited to an indirect reconstruction based on the established 3-D structure of both sub-units and their mutual position after association.

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Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

Biochemical Journal ◽

10.1042/bcj20190812 ◽

2019 ◽

Vol 476 (24) ◽

pp. 3835-3847 ◽

Cited By ~ 1

Author(s):

Aliyath Susmitha ◽

Kesavan Madhavan Nampoothiri ◽

Harsha Bajaj

Keyword(s):

Protein Engineering ◽

Cell Attachment ◽

Functional Characterization ◽

Protein Structures ◽

Structural Similarity ◽

Surface Proteins ◽

Sortase A ◽

Peptide Cleavage ◽

New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

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