Analysis of mutations and suppressors affecting interactions between the subunits of the HIV type 1 reverse transcriptase.

HIV-1 reverse transcriptase (RT) catalyzes the conversion of genomic RNA into cDNA. The enzyme is a heterodimer of p66 and p51 subunits, and the dimerization of these subunits is required for optimal enzyme activity. To analyze this process at the genetic level, we developed constructs that permit the detection of the interaction between these subunits in the yeast two-hybrid system. Genetic analysis of RT subdomains required for heterodimerization revealed that the fingers and palm of p66 were dispensable for p51 interaction. However, as little as a 26-amino acid deletion at the C terminus of p51 prevented dimerization with p66. A primer grip mutation, L234A, previously shown to inhibit RT dimerization by biochemical assays, also prevented RT dimerization in the yeast two-hybrid system. Second-site mutations that restored RT dimerization in yeast to the L234A parent were recovered in the tryptophan repeat region at the dimer interface and at the polymerase active site, suggesting the involvement of these sites in RT dimerization. In vitro binding experiments confirmed the effects of the L234A mutation and the suppressor mutations on the interaction of the two subunits. The RT two-hybrid assay should facilitate the extensive genetic analysis of RT dimerization and should make possible the rapid screening of potential inhibitors of this essential process.

HLA-DR polymorphism affects the interaction with CD4.

Major histocompatibility complex (MHC) class II molecules are highly polymorphic and bind peptides for presentation to CD4+ T cells. Functional and adhesion assays have shown that CD4 interacts with MHC class II molecules, leading to enhanced responses of CD4+ T cells after the activation of the CD4-associated tyrosine kinase p56lck. We have addressed the possible contribution of allelic polymorphism in the interaction between CD4 and MHC class II molecules. Using mouse DAP-3-transfected cells expressing different isotypes and allelic forms of the HLA-DR molecule, we have shown in a functional assay that a hierarchy exists in the ability of class II molecules to interact with CD4. Also, the study of DR4 subtypes minimized the potential contribution of polymorphic residues of the peptide-binding groove in the interaction with CD4. Chimeras between the DR4 or DR1 molecules, which interact efficiently with CD4, and DRw53, which interacts poorly, allowed the mapping of polymorphic residues between positions beta 180 and 189 that can exert a dramatic influence on the interaction with CD4.

Quantification of tertiary structural conservation despite primary sequence drift in the globin fold.

The globin family of protein structures was the first for which it was recognized that tertiary structure can be highly conserved even when primary sequences have diverged to a virtually undetectable level of similarity. This principle of structural inertia in molecular evolution is now evident for many other protein families. We have performed a systematic comparison of the sequences and structures of 6 representative hemoglobin subunits as diverse in origin as plants, clams, and humans. Our analysis is based on a 97-residue helical core in common to all 6 structures. Amino acid sequence identities range from 12.4% to 42.3% in pairwise comparisons, and, despite these variations, the maximal RMS deviation in alpha-carbon positions is 3.02 A. Overall, sequence similarity and structural deviation are significantly anticorrelated, with a correlation coefficient of -0.71, but for a set of structures having under 20% pairwise identity, this anticorrelation falls to -0.38, which emphasizes the weak connection between a specific sequence and the tertiary fold. There is substantial variability in structure outside the helical core, and functional characteristics of these globins also differ appreciably. Nevertheless, despite variations in detail that the sequence dissimilarities and functional differences imply, the core structures of these globins remain remarkably preserved.