Structural basis of the lysis clock in bacteriophages: the holin-antiholin interaction of phage lambda
EMSL Project ID
30460
Abstract
Bacteria are the most abundant organisms in the biosphere, and, after cell division, the second most common fate of bacterial cells is lysis by bacteriophage. For most bacteriophages, the duration of the infection cycle is governed by the function of a small membrane protein: the holin. The holin accumulates in the host membrane throughout the morphogenesis period, during which the progeny phage particles are assembled. At a programmed time, the holin proteins suddenly oligomerize to form large "holes" in the cytoplasmic membrane, which terminates macromolecular synthesis and allows the phage-encoded endolysin to attack the cell wall, resulting in lysis of the cell within seconds. The triggering time is encoded in the polypeptide sequence of the holin, which determines the interactions among holin protomers and, in some phages, with the antiholin, an antagonist of the holin. The interactions between holin proteins are so finely tuned that almost any amino acid substitution in the transmembrane domains of the holin accelerates or retards the triggering time and thus shortens or extends the infection cycle. Despite the fundamental character of this simplest of biological timing mechanisms, very little is known about its structural basis. Its elucidation may have further important ramifications in the understanding of such mechanisms in higher cells. Because the process of holin oligomerization appears to be governed by a subtle balance of interactions between individual transmembrane helices, we believe that its study fits well into EMSLs science theme of biological interactions and dynamics. Using EMSLs high-field solution NMR instruments, we propose to determine the structural basis for the mechanism of holin oligomerization. In a first stage, we will study the structure of a truncated holin protein that acts as an artificial inhibitor of hole formation. For this part of the project, we have already produced high-quality samples for NMR structure determination and obtained initial chemical shift assignments, but the NMR capabilities at our institution (600 MHz) are not sufficient for completing the study of these membrane protein samples. In a second part of the project, we will determine the structure of a mutant of full-length holin that does not oligomerize, and is thus still amenable to NMR study. On the basis of the NMR assignments and structure of these two proteins, we will characterize dimers that are formed between the full-length protein and the artificial or the natural inhibitor. The identification of dimerization interfaces, as well as the thermodynamic stability of dimers as measured by NMR should give detailed insight into holin oligomerization and its inhibition, some of which may also be extrapolated to other molecular clock mechanisms.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2008-10-01
End Date
2009-09-30
Status
Closed
Released Data Link
Team
Principal Investigator