Sandbox Reserved 196: Difference between revisions

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== Introduction ==
== Introduction ==


RNase B is a glycoprotein with N-linked carbohydrates. This sugar chain aids in the folding of the protein as well as well as cell to cell signaling<ref name="first">PMID:20301239</ref>. This plays an important role in tumor formation because it has been found that N-linked glycans recognized by the CD337 receptor on "natural killer cells" are mutated in tumor cells, stopping the death of these cells<ref name="second">http://en.wikipedia.org/wiki/Glycan#Functions_and_importance</ref>. RNase B is structurally the same as RNase A, however it has additional catalytic activity caused by the attachment of polysaccharrides at the <scene name='Sandbox_Reserved_196/Rbb_basic/8'>Asn-34</scene>. This small change allows RNase B to hydrolyze double-stranded RNA at ionic strengths where RNase A has no activity, showing that small changes in the active sites of very similar molecules can lead to totally new roles and activities <ref name="third">PMID:3680242</ref>. <scene name='Sandbox_Reserved_196/Rbb_basic/1'>(Return to original scene)</scene>
Ribonuclease B (RNase B) is a form of the enzyme RNase A that has an added glycoprotein with N-linked carbohydrates at <scene name='Sandbox_Reserved_196/Rbb_basic/8'>Asn-34</scene>. This added sugar chain aids in the folding of the protein as well as cell to cell signaling<ref name="first">Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME. ''Essentials of Glycobiology''. PMID:20301239</ref>. Glycoproteins play an important role in tumor formation because it has been found that N-linked glycans recognized by the CD337 receptor on "natural killer cells" are mutated in tumor cells, stopping their death<ref name="second">http://en.wikipedia.org/wiki/Glycan#Functions_and_importance</ref>. Other than the attachment of the polysaccharride, RNase B is structurally the same as RNase A, but the attachment allows for additional catalytic activity. This small change allows RNase B to hydrolyze double-stranded RNA at ionic strengths where RNase A has no activity, showing that small changes in the active sites of very similar molecules can lead to new roles and activities <ref name="third">PMID:3680242</ref>. <scene name='Sandbox_Reserved_196/Rbb_basic/1'>(Return to original scene)</scene>


== Structure and Biology of RNase B ==
== Structure and Biology of RNase B ==
[[Image:RNase B.jpg | thumb|left|RNase B]]
[[Image:RNase B.jpg | thumb|left|RNase B]]
RNase A and RNase B have identical primary structures; however, RNase B is bound to mannose carbohydrates. This glycosylation increases the kinetic stability of RNase B by 3 kj/mol compared to RNase A <ref name="fourth">PMID:10600722</ref>. Gycosylated, RNase B however, is not significantly different by NMR in protein <Structure load='1rbj' size='250' frame='true' align='right' caption='Ribonuclease B with a strand of DNA in active site' scene='Sandbox_Reserved_196/Secondary_structure/8' name ='1rbj' />conformation to RNase A <ref name="fifth">PMID:1322837</ref>.
Slight differences indclude changes in crystal packing. RNase B crystals have two slightly asymmetrical units. The crystals are dimeric with two separate molecules, I and II, which are linked by a salt bridge at Asp-121 and Arg-85. This linkage determines the orientation of the two molecules in relation to one another. Not only does a salt bridge link this dimer-type molecule, but other ions also interact via cross-linkage to stabilize the structure <ref name="third" />. 
The crystallization of RNase B provided the structure of the active site in which double stranded RNA is hydrolyzed.  The active site, a triangle formation of <scene name='Sandbox_Reserved_196/Secondary_structure/11' target='1rbj'>Lys-41, His-12, and His-119</scene> was shown to be the most intense active site and is found in both molecules I and II of RNase B.  In molecule II, the most drastic difference is the proximity of the active site to Lys-66, because ions can ligand to <scene name='Sandbox_Reserved_196/Secondary_structure/10' target='1rbj'>Lys-66, Arg-39 and Lys-1</scene>. Even though both active sights are close to identical, the two separate molecules are packed very differently from one another.  These active sights have been seen to deviate less from their “true” positions than those molecules in RNase A.  Shown in the image, the region of <scene name='Sandbox_Reserved_196/Rbb_basic/9' target='1rbb'>residues 15-23</scene> (in top applet) appear to have more flexibility, and upon looking at the structure could provide the opening for the active site. This catalytic site, with all the structures shown, has still not been an aid in providing the mechanism by which RNase performs its duty of hydrolyzing double stranded RNA <ref name="third" />.  <scene name='Sandbox_Reserved_196/Secondary_structure/8' target='1rbj'>(Return to original scene)</scene>


Crysallization of RNase A and RNase B has shown that these two enzymes are identical in their<Structure load='1rbj' size='250' frame='true' align='right' caption='Molecule II of Ribonuclease B with a strand of DNA in active site' scene='Sandbox_Reserved_196/Secondary_structure/8' name ='1rbj' /> primary structure and amino acid makeup; however, RNase B has a single glycosylation at the <scene name='Sandbox_Reserved_196/Rbb_basic/8'>Asn-34</scene> site that differentiates RNase B from RNase A.  RNase B has from five to nine mannose residues attached which can create a variance within RNase B molecules.  This glyosylation increases the kinetic stability of the RNase B by 3 kJ/mol<ref name="fourth">PMID:10600722</ref>. This addition to RNase B, however, does not significantly change the protein conformation from RNase A<ref name="fifth">PMID:1322837</ref>. 


Slight differences include changes in crystal packing, as the RNase B crystals have two slightly asymmetrical units. The crystals are dimeric with two separate molecules, I and II, which are linked by a salt bridge at Asp-121 and Arg-85. This linkage determines the orientation of the two molecules in relation to one another. Not only does a salt bridge link this dimer-type molecule, but other ions also interact via cross-linkage to stabilize the structure <ref name="third" />. 


The crystallization of RNase B, in complex with sequence DNA, provided the structure of the active site when bound to nucleic acids. The active site, composed of <scene name='Sandbox_Reserved_196/Secondary_structure/11' target='1rbj'>His-12, Lys-41 and His-119</scene> and found in both molecules I and II of RNase B is very similar to the active site of RNase A. A difference is the <scene name='Sandbox_Reserved_196/Rbb_basic/9' target='1rbb'>residues 15-23</scene> (in top applet), which are very flexible and can open up or close off the active site. Molecules I and II are slightly asymmetrical, and the most noticeable difference between the two is the position of the Lys-66. This residue, which is present in both molecules, is much closer to the active site in molecule II. This is important because ions bind to Lys-66, like the DNA, making them accessible to the active site. Even though the crystallization of the structure has been successful, it has not been an aid to providing the mechanism by which RNase B has the catalytic activity to hydrolyze double stranded RNA.<ref name="third" />.  <scene name='Sandbox_Reserved_196/Secondary_structure/8' target='1rbj'>(Return to original scene)</scene>
 
== References ==
== References ==
<references />
<references />

Revision as of 20:30, 14 April 2011

This Sandbox is Reserved from Feb 02, 2011, through Jul 31, 2011 for use by the Biochemistry II class at the Butler University at Indianapolis, IN USA taught by R. Jeremy Johnson. This reservation includes Sandbox Reserved 191 through Sandbox Reserved 200.
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3D picture of RNase B dimer

Drag the structure with the mouse to rotate
  Export Animated Image   Print3D

Introduction

Ribonuclease B (RNase B) is a form of the enzyme RNase A that has an added glycoprotein with N-linked carbohydrates at Asn-34. This added sugar chain aids in the folding of the protein as well as cell to cell signaling[1]. Glycoproteins play an important role in tumor formation because it has been found that N-linked glycans recognized by the CD337 receptor on "natural killer cells" are mutated in tumor cells, stopping their death[2]. Other than the attachment of the polysaccharride, RNase B is structurally the same as RNase A, but the attachment allows for additional catalytic activity. This small change allows RNase B to hydrolyze double-stranded RNA at ionic strengths where RNase A has no activity, showing that small changes in the active sites of very similar molecules can lead to new roles and activities [3]. (Return to original scene)

Structure and Biology of RNase B

File:RNase B.jpg
RNase B

Crysallization of RNase A and RNase B has shown that these two enzymes are identical in their

Molecule II of Ribonuclease B with a strand of DNA in active site

Drag the structure with the mouse to rotate
  Export Animated Image   Print3D

primary structure and amino acid makeup; however, RNase B has a single glycosylation at the Asn-34 site that differentiates RNase B from RNase A. RNase B has from five to nine mannose residues attached which can create a variance within RNase B molecules. This glyosylation increases the kinetic stability of the RNase B by 3 kJ/mol[4]. This addition to RNase B, however, does not significantly change the protein conformation from RNase A[5].

Slight differences include changes in crystal packing, as the RNase B crystals have two slightly asymmetrical units. The crystals are dimeric with two separate molecules, I and II, which are linked by a salt bridge at Asp-121 and Arg-85. This linkage determines the orientation of the two molecules in relation to one another. Not only does a salt bridge link this dimer-type molecule, but other ions also interact via cross-linkage to stabilize the structure [3].

The crystallization of RNase B, in complex with sequence DNA, provided the structure of the active site when bound to nucleic acids. The active site, composed of His-12, Lys-41 and His-119 and found in both molecules I and II of RNase B is very similar to the active site of RNase A. A difference is the residues 15-23 (in top applet), which are very flexible and can open up or close off the active site. Molecules I and II are slightly asymmetrical, and the most noticeable difference between the two is the position of the Lys-66. This residue, which is present in both molecules, is much closer to the active site in molecule II. This is important because ions bind to Lys-66, like the DNA, making them accessible to the active site. Even though the crystallization of the structure has been successful, it has not been an aid to providing the mechanism by which RNase B has the catalytic activity to hydrolyze double stranded RNA.[3]. (Return to original scene)

References

  1. ↑ Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME. Essentials of Glycobiology. PMID:20301239
  2. ↑ https://en.wikipedia.org/wiki/Glycan#Functions_and_importance
  3. ↑ 3.0 3.1 3.2 Williams RL, Greene SM, McPherson A. The crystal structure of ribonuclease B at 2.5-A resolution. J Biol Chem. 1987 Nov 25;262(33):16020-31. PMID:3680242
  4. ↑ Imperiali B, O'Connor SE. Effect of N-linked glycosylation on glycopeptide and glycoprotein structure. Curr Opin Chem Biol. 1999 Dec;3(6):643-9. PMID:10600722
  5. ↑ Joao HC, Scragg IG, Dwek RA. Effects of glycosylation on protein conformation and amide proton exchange rates in RNase B. FEBS Lett. 1992 Aug 3;307(3):343-6. PMID:1322837

Additional Resources

  1. https://www.pdb.org/pdb/explore/explore.do?structureId=1RBJ
  2. https://www.pdb.org/pdb/explore/explore.do?structureId=1RBB
  3. Ribonuclease
  4. User:R. Jeremy Johnson
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