Student Projects for UMass Chemistry 423 Spring 2012-10

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Leadzyme, 1nuv

Leadzyme 1nuv

Introduction

Leadzyme is a ribozyme, created in vitro through artificial selection. In the presence of lead, leadzyme will bind and cut RNA. The cleavage reaction is a two step process. First the ribozyme uses the bound lead ion to cleave the phosphodiester backbone of the substrate strand. This produces both a free 5' hydroxyl group and a 2',3' cyclic phosphodiester. The cyclic phosphate is then hydrolyzed yielding a 3'-phosphate. Leadzyme has two different states which it transitions between.
The first is the . This state happens when lead binds causing C23 to align with the scissile G24 phosphodiester bond. In the ground state there is no ion pulling these residues so they are farther apart. It is believed that may act as a competitive inhibitors for this ribozyme. Strontium is hydrated and binds to ribonucleotides 23,26 and 43-45. The interactions formed are hydrogen bonds via the water and the bases. The strontium binding sites overlap with the lead binding sites, leading to the belief that it may act as a competitive inhibitor. is also a known inhibitor of this ribozyme, but it is believed to be an allosteric inhibitor. It has the different binding sites across bases 19-20,24-25,29-30,39-43,47. When it binds it causes conformational changes within the ribozyme that disrupt its normal cleavage functions and return it to its ground state conformation. between G42 and G43 and the substrate strand of RNA which constrains the trinucleotide bulge. Without this interaction, the ribozyme relaxes into its precatalytic state.
Leadzyme is thought to have similarities with 5s subunit of the rRNA of ribosomes. This has lead people to the hypothesis that lead poisoning stems from ribosomal malfunction due to the presence of lead ions. There are D-loops of the rRNA have similar sequences and when exposed to lead can have cleavage activity. If the concentration of lead in the body is enough to interact with a significant number of ribosomes they could begin to cleave necessary mRNAs, which would be detrimental to the body.



Overall Structure

Insert caption here

Leadzyme is made of a RNA duplexe shown here with nucleotides 18-30 shown in orange and nucleotides 39-49 shown in blue. The duplex contains two The C23 to A45 is shown in black and A25 to G44 is shown in blue, which form a 3 nucleotide bulge. Divalent ions bridge the complementary strands by binding tandem purines from one strand to the major groove. The scissile phosphodiester bond fits at at the junction between the duplex and the bulge. The nucleotides forming the bulge have been shown to be relatively mobile and 2 significantly different confirmations have been documented. The main difference between the confirmations is that in one the all nucleotides in the bulge point away from the interior of the duplex while in the other confirmation the A in the bulge points inward.










Binding Interactions

Special features of leadzyme.

Pb2+ in leadzyme cleaves the RNA by cleaving the substrate’s backbone and producing a 2’,3’-cyclic phosphodiester and a 5’-hydroxyl. This first step is common among many ribozymes, however, the 2’,3’-cyclic phosphodiester is hydrolyzed to produce a 3’-phosphodiester.

Leadzyme requires Pb2+ to cleave RNA and is inhibited by Mg2+, through competitive inhibition. A possible suggestion for Mg2+ inhibition may have to do with the Mg(H2O)6 which bind in the major groove at the G39, G30 and A29 nucleotides at one location (, in both the ground state and pre-catalytic state), at the U47, G20, and G19 nucleotides from another location (, from both states), and at the U41, G42, G43, G24, and A25 nucleotides in the ground state (). This last interaction is particularly important since it “ligates” the , which inhibits the activity of the leadzyme. Since this type of interaction occurs only in the ground state, it is thought to stabilize the ground state and thus prevent catalysis of RNA cleavage. The Mg2+ "pulls in" the trinucleotide bulge by hydrogen bonding withN7 and O6 in G42 and O4 in U41 with a "sphere" of six water molecules attached to the magnesium ion. It is worth noting that because of the competition between metal ions in leadzyme, if lead is added to the ground state form of leadzyme with bound Mg2+ at site II, the structure may change from ground state to pre-catalytic by interrupting the hydrogen bonding/bridging between the two tandem purines (G42 & G43) and A25 and G24, thus relaxing the trinucleotide bulge.There are some differences in the types of binding involved in sites II than in sites I & III. For example. In site II, O6 at the G43 nucleotide shares an H2O with O6 of G42 rather than hydrogen bonding with a separate water molecule.

Another ion of interest is Sr2+, which binds similarly as Pb2+ but has different catalytic effects due to electronic differences. Similar bonding occurs because of similar ionic radii (Sr2+=1.12A; Pb2+=1.13A). Although there are multiple major Sr2+ binding sites, one particularly important site in the pre-catalytic form interacts with , which is located in the Scissile Bond region. At this bonding site, Sr2+ ligates N1 of A45 and non bridging phosphate of A25. This bonding dramatically changes the conformation of the phosphodiester backbone (in the presence of Mg2+ and Sr2). In the ground state , which helps stabilize the ground state. As with Mg2+, there is a H2O sphere around Sr2+ and this allows hydrogen bonding between N7 of G23, N6 of A 45, O6 of G44, and a "nonbriding phosphate oxygen of G43". Also, it is important to notice that the binding of Sr2+ is adjacent to Site II of Mg2+ in the ground state and has very little affect on the binding of Mg2+ at this site. It is important that metal ions such as Sr2+ and Mg2+ inhibit leadzyme since without this "stable" crystal structure, leadzyme would continuously cleave RNA, including itself.

Because of such interactions, leadzyme in the presence of only Sr2+ does not show significant cleavage, where leadzyme in the presence of Sr2+ and Pb2+ shows reduced cleavage in comparison to leadzyme and only Pb2+.

Additional Features

Special features of leadzyme.

The is dependent on the inclusion of lead for catalytic effect: no other ion gives the same result. The particular area of interest for the cleavage activity occurs at a .

The catalytic cleavage depends on the presence of lead (II) in solution, and is further catalyzed by the addition of a second metal ion, particularly magnesium (II) or lanthanide ions. Strontium (II) appears to occupy the same positions as lead (II), and is used for imaging purposes, but does not have any catalytic action.

Studies show that equimolar solutions of neodymium (III) and lead (II) maximize this effect, suggesting that neodymium acts in concert with lead, as an acid catalyst. The site at which the RNA is cleaved remains unchanged.[1] [2] As shown by Wedekind and McKay, the lead ion may interact with the major grove of the base pairs near or in the loop area, which the secondary ion is located a few residues to either side. Both metals hydrogen bond through their hexaaquo coordination spheres.

The actions of both the lead ion and the secondary metal ion appears to be highly pH dependent. Though variation in the pKa values changing the protonated forms of functional groups are more frequently a concern for amino acids for which more pKa's are within the range of biological pH, the leadzyme provides an example where the rate shifts are also associated strongly with pH.

The leadzyme [3] rate demonstrates a strong dependence on pH. The pKa values for the residues at the active site loop are affected by their environment. Activity has been found to be highly dependent on pH near to the biological range, with the maximum rate for lead-only solution occuring at 7, and for lead and magnesium is solution at 7.2. The rate constant increases linearly, increasing almost 100-fold from pH of 5.5 to 7.2. [4]

The shows residues with pKa < 3.1, pKa of 4-5 and pKa > 5. The pH closest to the biological range, residue A45, is an adenine that participates in coordination of the lead at one of three identified lead binding sites. This adenine is also part of the only non-Watson-Crick pair near the active site. The loop connecting the two strands consists of adenines with pKa around 3.5.[5] The does not exhibit an considerable increase in stability at lower pH as experienced by some similar sequences,[6] which would be consistent with the pH instead changing the lead binding capability.

Credits

Introduction - Jeffrey Salemi

Overall Structure - Adam Ramey

Binding Interactions- Nicholas Vecchiarello

Additional Features - Tom Foley

References

  1. Ohmichi, T., Sugimoto, N. (1996) FEBS Letters 393, 97-100.
  2. Ohmichi T, Sugimoto N. Role of Nd3+ and Pb2+ on the RNA cleavage reaction by a small ribozyme. Biochemistry. 1997 Mar 25;36(12):3514-21. PMID:9132001 doi:10.1021/bi962030d
  3. Wedekind JE, McKay DB. Crystal structure of the leadzyme at 1.8 A resolution: metal ion binding and the implications for catalytic mechanism and allo site ion regulation. Biochemistry. 2003 Aug 19;42(32):9554-63. PMID:12911297 doi:http://dx.doi.org/10.1021/bi0300783
  4. Pan T, Dichtl B, Uhlenbeck OC. Properties of an in vitro selected Pb2+ cleavage motif. Biochemistry. 1994 Aug 16;33(32):9561-5. PMID:8068631
  5. Legault, P., Pardi, A. (1997) J. Am. Chem. Soc. 119, 6621-6628
  6. Chen G, Kennedy SD, Turner DH. A CA(+) pair adjacent to a sheared GA or AA pair stabilizes size-symmetric RNA internal loops. Biochemistry. 2009 Jun 23;48(24):5738-52. PMID:19485416 doi:10.1021/bi8019405

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