Abstract
The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity.
Highlights
The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is a small nucleic acid binding protein that possesses a N-terminal basic domain and two zinc fingers connected by a short linker (Figure 1A)
Zinc fingers are thought to play a major part in nucleic acid destabilization, fast binding, and dynamic restriction, while the basic N-terminal part is mainly responsible for the nucleic acid aggregation activity [1,2,3,11,14,15,16,17,18,19]
15N relaxation studies of free NC show that the two zinc fingers move relatively to each other around a hinge of small size (G35 and perhaps K34)
Summary
The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is a small nucleic acid binding protein that possesses a N-terminal basic domain and two zinc fingers connected by a short linker (Figure 1A). The NC domain, under its various forms (Gag, NCp15, NCp9, NCp7) plays numerous roles during the replication cycle of the virus [1,2,3] Among these forms, NCp7 (named NC in this report), via its nucleic acid chaperone activity [1,4,5], is thought to facilitate the strand transfer processes occurring during reverse transcription [6,7,8,9]. Zinc fingers are thought to play a major part in nucleic acid destabilization, fast binding, and dynamic restriction, while the basic N-terminal part is mainly responsible for the nucleic acid aggregation activity [1,2,3,11,14,15,16,17,18,19]
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