Synonymous mutations and ribosome stalling can lead to altered folding pathways and distinct minima

Abstract

How can we understand a case in which a given amino acid sequence folds into structurally and functionally distinct molecules? Synonymous single-nucleotide polymorphisms in the MDR1 (multidrug resistance 1 or ABCB1) gene involving frequent-to-rare codon substitutions lead to identical protein sequences. Remarkably, these alternative sequences give a protein product with similar but different structures and functions. Here, we propose that long-enough ribosomal pause time scales may lead to alternate folding pathways and distinct minima on the folding free energy surface. While the conformational and functional differences between the native and alternate states may be minor, the MDR1 case illustrates that the barriers may nevertheless constitute sufficiently high hurdles in physiological time scales, leading to kinetically trapped states with altered structures and functions. Different folding pathways leading to conformationally similar trapped states may be due to swapping of (fairly symmetric) segments. Domain swapping is more likely in the no-pause case in which the chain elongates and folds simultaneously; on the other hand, sufficiently long pause times between such segments may be expected to lessen the chances of swapping events. Here, we review the literature in this light.

Figure 1

Simplified folding free energy landscape to illustrate two types of protein folding pattern scenarios. At the single molecule level, the native protein conformation is more favorable thermodynamically than the altered conformation due to synonymous mutations (Figure 1A). However, the folding free energy shifts toward favoring the haplotype conformation at high concentration with a portion of the conformation changed significantly due to inter-molecular association. The sizable barrier in Figure 1A reflects the involvement of a significant conformational change. Figure 1B illustrates the second type of folding pattern. At the bottom of the folding funnel, the landscape is rugged with many local minima, each representing a similar but distinct core structure. The intermediate barrier in the Figure 1B is to emphasize that there is only a minor conformational change when moving from one local minimum to the other. Unlike the first (amyloid) folding pattern, here the folding free energy landscape will not change since it does not involve inter-molecular interactions. The final folded conformation is mainly controlled by folding kinetics: a different folding pathway leads to a different conformation.

Figure 2

A simple scheme to illustrate the origin of a minor conformational change via a ribosome stalling effect. Along a sequence, say at an arbitrary position S₁, fragment A preceding S₁ is a slow folder and the fragment B following S₁ folds faster than fragment A. Also, fragment A has two competing conformations, A₁ and A₂ with A₂ more stable than A₁ by itself but A₁ becomes more favorable in the presence of fragment B. Let us assume that the folding of the nascent chain is independent. Then the folding landscape will be exactly the same for both the wild sequence (W) and the sequence (S) with a synonymous mutation at S₁ since they have the same amino acid sequence. The co-translational folding pathway is expected to be identical up to the S₁ position. In Figure 2A the folding pathways are drawn as step-by-step arrows on the simplified folding funnel surface. Without a pause at S₁, fragment B folds before fragment A; then fragment A folds on fragment B with an A₁ conformation. On the other hand, for the synonymous mutation at S₁ case, the pause enables A₂ to fold first and fragment B follows. The folding branches due to a pause in a sequential folding eventually lead to the bottom of funnel with minor conformational change between them. Figure 2B provides a diagram of the two folding scenarios.