Investigation of Phycobilisome Subunit Interaction Interfaces by Coupled Cross-linking and Mass Spectrometry

Ofir Tal,Beny Trabelcy,Yoram Gerchman,Noam Adir

doi:10.1074/jbc.m114.595942

Abstract

The phycobilisome (PBS) is an extremely large light-harvesting complex, common in cyanobacteria and red algae, composed of rods and core substructures. These substructures are assembled from chromophore-bearing phycocyanin and allophycocyanin subunits, nonpigmented linker proteins and in some cases additional subunits. To date, despite the determination of crystal structures of isolated PBS components, critical questions regarding the interaction and energy flow between rods and core are still unresolved. Additionally, the arrangement of minor PBS components located inside the core cylinders is unknown. Different models of the general architecture of the PBS have been proposed, based on low resolution images from electron microscopy or high resolution crystal structures of isolated components. This work presents a model of the assembly of the rods onto the core arrangement and for the positions of inner core components, based on cross-linking and mass spectrometry analysis of isolated, functional intact Thermosynechococcus vulcanus PBS, as well as functional cross-linked adducts. The experimental results were utilized to predict potential docking interactions of different protein pairs. Combining modeling and cross-linking results, we identify specific interactions within the PBS subcomponents that enable us to suggest possible functional interactions between the chromophores of the rods and the core and improve our understanding of the assembly, structure, and function of PBS.

Highlights

The phycobilisome is assembled from many subunits, but the entire structure has not been determined
Functional T. vulcanus PBSs (Tv-PBS) complexes were isolated in a high concentration of phosphate buffer followed by sucrose gradient centrifugation (20)
Tv-PBS energy transfer to APC was monitored by excitation of PC at 540 nm, with emission from APC at 660 – 680 nm and minimal fluorescence at 645– 655 nm

Summary

Background

The phycobilisome is assembled from many subunits, but the entire structure has not been determined. A similar physical gap exists within the PBS complex itself: energy absorbed within the rods has to be efficiently transferred to the core (containing APC and the terminal emitter minor components ApcE and ApcD (8)). It is possible that the PBS are tightly packed within the interthylakoid membrane stromal space (19) and that rods could associate with more than one PBS core, making all chromophores efficient in energy transfer This organization would, place the position of the rod aperture (where the LPs are located) in-between complexes. We present the results of the integration of chemical cross-linking under native conditions and MS, combined with existing high resolution crystallographic structures of isolated subunits and molecular modeling to reach conclusions about the architecture of the complex. On the basis of these results, we were able to propose structural arrangement of PBS subcomponents inside the core and between the rods and core elements

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION