PRESUPUESTO DE INGRESOS Y GASTOS DE LA INSTITUCIÓN EDUCATIVA REPÚBLICA DE VENEZUELA
13. PLAN DE ACCIÓN INSTITUCIÓN EDUCATIVA REPÚBLICA DE VENEZUELA
This diagram was constructed using the program TURBO-FRODO (Cambillau et ai,
1 996) from the PDB entry CRB 1 (Cowan et aI. , 1 993). a-Helices are coloured red,
1.3. 1.2 Structure of CRBP I
1 -28
The first crystals of CRBP were grown in 1 98 1 (Newcomer et al., 1 98 1 ), and the
structure of CRBP in complex with all-trans retinol was solved to 2 . 1 A resolution in 1 993 (Cowan et al., 1 993). The large time period ( 1 2 years) between obtaining crystals
and publishing the structure, indicates that this structure was exceedingly difficult to determine. Crystals were grown by hanging drop vapour diffusion of rat liver CRBP protein. The crystals were orthorhombic with a space group P2j212j. The structure was solved using molecular replacement from P2 myelin protein, and the final R-factor was 1 8.8 % .
The basic structure o f CRBP i s a single domain orthogonal p-barrel (Figure 1 5). Although primary sequence identity between iLBP family members is fairly low - as little as 1 2 % identity between CRBP I and an insect fatty acid binding protein - the basic structural framework is highly conserved within the family. The structure consists of 1 0 anti-parallel f3-strands organised into 2 anti-parallel p-sheets which are folded over to create the 'barrel' . Each strand is connected to the next by a short reverse turn, except strands A and B which are connected by a helix-turn-helix motif.
1.3.1 .3 Ligand Binding
The ligand fits into the centre of the barrel, lying along the barrel axis, with the functional group in the core of the protein (Figure 1 .5). Interactions in the form of hydrogen bonds, salt links and hydrophobic interactions are made both with internal protein side chains (from sheets and helices), and with buried solvent molecules. There is no connection between the internal closed cavity and the external solvent.
There are usually 2 entrances to the internal cavity created by an orthogonal barrel structure. However in CRBP I, both of these are closed off - one by a-helices and the other by side chain residues. This raises an interesting question as to how the ligand gains entry to and is released from the protein. The structures of the apo- and the holo proteins show very few differences (Winter et al., 1 993), and it is not obviously apparent
how retinoids enter into the binding cavity. A current theory is that time dependent fluctuations in the conformation of the structure exist in solution. It is likely that one or more of these conformations may be more open which may allow ligand access. A candidate for the flexible part of the structure which could facilitate access is the helix turn-helix motif between sheets A and B, functioning as a 'helical cap' (Jamison et al.,
1 -29
1 994). Favourable van der Waals contacts between the retinoid J3-ionone ring and the helices exist in the holoprotein which may stabilise this structure. However in the absence of ligand, and without these interactions, the helical cap may be able to move enough to allow the ligand to enter. A similar process has been described for two lipases
(Brzozowski et al., 1 99 1 ; Tilbeurgh et al., 1 993).
An examination of the tertiary structures of apo- and holo - CRBP I gives a structural
basis for the observed differences in affinity for all-trans retinol and retinal. There are a number of general interactions between the binding protein and the ligand, which would be expected to occur whether retinol or retinal was the ligand. A positive charge on the side chain of lysine 40 interacts with the isoprene tail of the retinoid. There are eight water molecules in the internal cavity which participate in hydrogen bonding and create a cavity with a shape complementary to the ligand. However, there is one interaction at the tail end of the retinoid which occurs with all-trans retinol as the ligand but not with retinal (Figure 1 .6). The carbonyl oxygen on the side chain of glutamine 1 08 (GIn 1 08) forms a hydrogen-bond with the alcohol moiety of retinol. At the same time, there is a stabilising interaction between an amino group hydrogen (Gin 1 08) and the phenylalanine ring of phenylalanine 4 (phe 4). When retinal is the ligand, the most likely situation is one where the same favourable electrostatic interaction between Phe 4 and Gin 1 08 occurs. This leaves no hydrogen donors in the proximity which are free to form an interaction with the oxygen from the aldehyde carbonyl group of retinal. Alternatively, GIn 1 08 could rotate, providing a hydrogen donor from the amino side-chain which would stabilise the binding of retinal. The hydrogen bonding geometry between GIn 1 08 and the carbonyl oxygen of retinal adopted in this scenario is sub-optimal, and this interaction would also erase the favourable electrostatic interaction with Phe 4. These observations largely account for the different dissociatio n constants observed for CRBP I binding to all-trans retinol and all-trans retinal.
The inability of CRBP I to bind retinoic acid, and eis-isomers of retinol and retinal may
be explained fairly straightforwardly. The binding of retinoic acid would introduce another negative charge into the barrel, leaving no space in the cavity for extra water molecules for solvation. This is considered very energetically unfavourable. The overall shape of the 9-eis or l l -eis isomers of retinal and retinol (Figure 1 . 1 ) compared to the all-trans conformation is such that it may be sterically impossible for the molecule to gain access to the highly restricted site, and for the molecule to fit in the highly defined site if it could gain access.
Phe 4 Phe 4