User:Christian Fjeld/Sandbox 1

General Description

is a 97 kDa, class IA aminoacyl-tRNA synthetase (ARS) that catalyzes the ligation of leucine with tRNA^leu in an ATP dependent mechanism. ARS are essential to all life as they charge amino acids onto cognate tRNAs in preparation for protein translation. This is step is a potential source of error in interpreting the genetic code as mischarged tRNAs will not be recognized during protein synthesis and could lead to nonfunctional proteins. As such, ARS have a high specificity for both tRNA and amino acids and contain an editing domain capable of hydrolyzing mischarged tRNA.

LARS is a cytoplasmic enzyme that is found as part of the multisynthetase complex in eukaryotes^[1]. The multi-synthetase complex contains glutamylprolyl-tRNA synthetase (EPRS), isoleucyl (IARS), leucyl (LARS), glutaminyl (GARS), methionyl (MARS), lysyl (KARS), arginyl (RARS), and aspartyl (DARS) tRNA synthetases as well as p18, p38 and p43^[2].

Multisynthetase complexes have also been seen in some archaea such as Thermococcus kodakarensis although the composition of the complex is not the same as eukaryotes^[3]. LARS has been shown to be involved with the mTOR pathways as a sensor of leucine levels within the cell^[4].

Disease

Mutations in LARS2, the mitochondrial version of the enzyme, have been linked to Perrault syndrome characterized by premature ovarian failure in females and progressive hearing loss in both sexes^[5]

Structure

Catalytic Domain

The is responsible for the two step process of charging leucine on to tRNA^leu. First, ATP and leucine are bound and AMP is transfered to the backbone carboxylic acid of leucine with the release of a pyrophosphate. Second, tRNA^leu is bound with the leucyl adenylate and leucine is transfered to either the 2' OH of the 3' terminal adenine with the release of AMP^[6].

LARS, like all class I synthetases, is characterized by a Rossmann-fold catalytic domain with a central parallel β-sheet with α-helices on both faces. The active site catalyzes both the formation of the leucyl adenylate intermediate and the subsequent charging of leucine onto the terminal acceptor arm of the tRNA^[7]. The binding pocket accommodates leucine as well as smaller amino acids such as isoleucine and valine. This allows tRNA^leu to be mischarged with noncognate amino acids and must be corrected to ensure translational fidelity^[8].

Editing Domain

The of LARS is responsible for hydrolysing mischarged tRNA, such as ile-tRNA^leu, and releasing it for a subsequent round of aminoacylation. This editing function allows cells to achieve a tRNA charging error rate of less than 1:10,000^[9]. The domain itself is an β sandwich connected to the catalytic domain by a flexible loop that allows a hinge action to bind aminoacylated tRNAs for error checking.

The binding pocket of the editing domain acts as a reciprocal sieve to the catalytic domain. The catalytic domain cannot distinguish between amino acids that are similar to, or smaller than leucine. The binding pocket of the editing site can accommodate these similar or smaller amino acids but excludes leucine due to a steric clash with a highly conserved theronine^[10]. Together, the catalytic site discriminates against larger or hydrophobically different amino acids and the editing site hydrolyzes smaller, structurally similar amino acids producing a very low error rate^[11].

Anticodon Binding Domain

The is essential for the fidelity of ARSs. However, there are 6 anticodons in E. coli that correspond to leucine including AAU, CUG, and GUG, so how does the enzyme recognize so many different tRNAs? This is accomplished by recognition of the D-loop by the enzyme rather than the actual anticodon. The anticodon binding domain interacts with , 12-16 and 22-26, with U16 being essential for recognition. U16 specifically interacts with Arg718 and Lys711 within helix 4 forming hydrogen bonds.

The anticodon bindind domain is an all α helical domain made up of 8 helices in a bundle. Helices 3-5 form the tRNA recognition surface and are responsible for interaction with the D-loop of the incoming tRNA.

Zinc Binding Domain

The

Leucine Specificity Domain

The , specifically the KMSKS loop, plays a major role in catalysis in many species. The domain is highly variable between species but the KMSKS loop is highly conserved^[12]. In catalysis the domain functions by closing over the 3' terminal adenine of the tRNA, closing the catalytic site and allowing catalysis to take place.

In E.coli the leucine-specific domain is composed of 2 3-strand antiparallel β-sheets and pivots on a flexible loop. The KMSKS loop spans residues 619-623 and can contact A76 of the tRNA within the catalytic site^[13]. There is also a β-hairpin in E. coli that contacts bases 10 and 27 of the tRNA^[14].

Evolutionary Conservation

LARS is essential for protein synthesis and as such is necessary for all cellular life and present in all three kingdoms of life. Eukaryal and archaeal LARS are similar and both are structurally different from bacterial LARS. Most of the differences occur in tRNA recognition sites while the core of the catalytic and editing domains are highly conserved. The differences between bacterial and archaeal tRNA, most notably the truncated variable arm in archaea, begin to explain the structural changes that are seen in the evolution of the enzyme^[15].

A shows that there is a high degree of conservation in LARS, especially within the catalytic domain. The anticodon binding domain shows a higher variation. This is to be expected as there is a larger amount of variation in codon usage, most notable between bacteria and archaea/eukarya.