CAPÍTULO IV. RESULTADOS Y DISCUSIÓN
4.1 Caracterizar el comportamiento de los hogares con respecto al manejo de residuos
There are 3 examples of crossed-ridge packings, a 3x4, a 4-4/4x4 and a 4x4. The 4-4/4x4 packing occurs in 6tmn 11-12 and is similar to those described previously
Ridge-groove model o f helix-helix packing
Figure 4.17: 4x4 packing
6tmn 2-5 has 4x4 packing and an interhelical angle {Q) of 95.8°. This angle
lies between the 1-4 = 22.5°, 13.4°) and 4-4 = 39.6°) packing regions
which is reflected by the interface. Tyr 83 is at the centre of the interfaces on helix 2, adjacent to another Tyr (84), a regular ridge-groove interaction could not possibly occur as these residues are far too large (Figures 4.17 and 4.19). Helix 5 also has 2 aromatic residues on its interface, Tyr 179 and Phe 172, disrupting the packing further. This packing was defined as a 4x4 by Chothia et a l (1981) but can equally be considered a distorted 1-4 with extra contacts.
One 3x4 packing was observed (Figure 4.18) in Figure 4.18: 3x4 packing 1116 8-10. The interaction angle (Q = 169.5°) lay
outside the ranges for any of the ridge-groove packing class. This packing can be described as a distortion of
4-4 packing; In 4-4 packing when observing the
interface plane, the i±3 and the i±A ridges on opposite faces lie roughly perpendicular to each other. This 3x4 packing resulted because the i±3 ridge was more prominent than the accompanying i±4 ridge.
6TMN 2-5
1116
8-10
0
/
0
---"
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4.3.7Av N ot assignedOnly one helix interaction could not be assigned 61yz 2-6 (£2= 117.6), the packing was highly irregular and seemed to be a mixture of 3-4 and 1x4.
(a)
Helix 5
Helix 2
Figures (a) and (c) show the van der Waals surfaces of the interacting helix pair, with helix 2 contoured in yellow and helix 5 contoured in red. Figures (b) and (d) show the interacting surface of the back "helix" in the same orientation as the preceeding figure ((a) and (c) resectively) with the interacting side chains highlighted in green.
Ridge-groove model o f helix-helix packing
4 .4 D i s c u s s i o n
In this chapter a comparison of the current models of helix packing was undertaken, followed by the manual assignment of the helix packing classes in 11 proteins. Although the packing classes of the helix pairs within 10 of these proteins had been previously assigned, several discrepancies between the reported assignments and those predicted by a preliminary algorithm led to their re-evaluation. Finally, the packing in only 36 helix pairs was considered, as they were the only helix pairs to fit our criteria of highly interacting helices (at least 7 interhelical residue-residue contacts were required with a delta_SA > 150Â^).
As an extension of previous models, novel packing classes for the more irregular packing motifs were defined: 4-4*, 4-4/4x4 or 4x4 for irregular 4-4 type packings and a non-classic ridge-groove class. This allowed a clearer and a more thorough classification, enabling the packing classes to be determined in 4 interfaces where previous assignment could not be or was not attempted. In addition, 8 of the new manual assignments differed from those previously reported. Several of the discrepancies are due to opinion on how distorted from 4-4 packing the interface is. However, some were due to slight differences in the proteins used, indicating that care should be taken in assuming that helix packing in homologous proteins is identical
Figure 4.19 shows the summary of the classifications. Those interactions that match the ridge-groove model are shown above the x-axis and those that do not lie below. It can be seen quite clearly that regular ridge-groove packings have interhelical interaction angles that lie within the expected regions, and in this respect the ridge-groove model is a good one. However, only 56% of the interactions fit this model. The figure also shows that non-ridge-groove packings are found quite
commonly within the regions where regular ridge-groove packings would be expected,
although they often lie on the outskirts of these regions.
Although irregular packings often have interhelical angles which correspond to the regular ridge-groove regions, they also lie between these regions. Their contact patterns reflect the mixture of the surrounding packing classes. That these packings lie between the "allowed" regions suggests the current models are too stringent. The
3 -- 2 - (0 C o
I
oI
E D z -3 -- -4 -- -5 ■■ I n t e r a c t i o n a n g l e iN o n classic 3-4 I C la ssic 3 - 4 I c i a s s i c 1-4 I N o n -C lassic 4-4 IC Iassic 4-4 14x4 I3x4 U nassigncd ■allow ed 3-4 'a llo w e d 4-4 'a llo w e d 1-4Figure 4.20 show s a summary o f the packing classes observed in the data set.
The bars above the x-axis indicate those packings that are ridge-groove, whilst those below the x-axis are those interactions in disagreement with the ridge-groove model.
Ridge-groove model o f helix-helix packing
models assume that the interface residues are regular and of similar size and as this is not the case helix pairs do not comply with these simple packing rules.
Ridge-groove is a good and simple model for the regular 4-4 and 1-4 packing although, even within the 4-4 classification, the interfaces vary considerably. Sub classifications would be necessary in the 4-4 packings to cluster the interactions more tightly, as current assignments are not adequate to find sequence patterns. The knobs into holes model is a better description for those interactions with interaction angles (H) found in the 3-4 regions.
There are several problems with the current method of assigning packing classes. It is time-consuming as it is dependent on the analysis of helix interaction diagrams and 3D representations of helix interfaces. It relies on a visual assessment of prominent ridges, which for the more complicated surfaces can be a matter of opinion. Although, the packing class is sometimes very clear, this is not the case for the more complicated packings.
This type of helix packing classification would be difficult to perform ^ computationally. The next chapter will describe how distance and contact maps were used to search a database of protein structures to identify and cluster a range of helix packing motifs.
Ch a p t e r 5
A
u t o m a t ic a l l y c l u s t e r in g h e l ix-
h e l ix p a c k in g sAll previous classifications of helix packing have been manual, but with 882 helix pairs in the 2.5Â non-homologous data set, an automatic method of classification was required. As shown in Chapters 3 and 4, helix pairs with very similar interaction angles can pack together very differently, with a variety of interhelical distances and residue packing patterns. It follows that the interhelical angle alone would be inadequate for distinguishing distinct packings types.
Results from Chapters 3 and 4 also suggest that the residue packing at helix interfaces seems as much a function of side-chain character as the intercalation of "knobs' on regular cylinders, suggesting that a sequence template approach may indeed
be viable. Distinguishing between regular and irregular interfaces might be
particularly important when differentiating sequence patterns.
When assigning the helix packing classes manually (in Chapter 4), it was the helix interaction diagrams that made the classification possible. There is no existing method to compare helix interaction diagrams computationally and there was no obvious method to do so. What was required was another 2D representation of the interface that retained the information content. Contact maps and distance matrices were chosen as a simple representation of helix interactions. Using the previous analyses from Chapter 4, initial inspection of the contact maps for 3-4 and 4-4 packings suggested that helix packing motifs could be easily distinguished.
This chapter starts with a review of general structural comparison methods that use the contact map/distance matrix approach, that could be applied to distinguish different residue packings at helix-helix interfaces. Both contact map and distance matrix approaches were used to automatically cluster homologous helix pairs in a set of globin structures. Having determined the optimal parameters, the method was used to determine the similarity of the packing of helix pairs in the Chothia data set of
Automatically clustering helix-helix packings
proteins, whose packing classes had been manually assigned. Some of the helix pairs clustered as expected, but there were several failures. This led to further reassessment of the Chothia classes of helix packing.
5.1 St r u c t u r a l c o m p a r i s o n m e t h o d s
Most of the current methods that are used to structurally compare protein domains and fragments (eg. SSAP, Taylor & Orengo, 1989a,b; Orengo et al., 1992; Orengo & Taylor, 1991) are designed to pull out gross topology equivalences and compare protein backbone conformations rather than side chain packing information. However, as there is a plethora of well documented structural comparison methods, it is likely one could be easily adapted to distinguish helix packing types. The problem is two fold, first a reliable alignment of equivalent residues has to be found, then a sensitive scoring scheme has to be established.