Nanomechanical and Thermophoretic Analyses of the Nucleotide-Dependent Interactions between the AAA⁺ Subunits of Magnesium Chelatase

Abstract

In chlorophyll biosynthesis, the magnesium chelatase enzyme complex catalyzes the insertion of a Mg2+ ion into protoporphyrin IX. Prior to this event, two of the three subunits, the AAA+ proteins ChlI and ChlD, form a ChlID–MgATP complex. We used microscale thermophoresis to directly determine dissociation constants for the I-D subunits from Synechocystis, and to show that the formation of a ChlID–MgADP complex, mediated by the arginine finger and the sensor II domain on ChlD, is necessary for the assembly of the catalytically active ChlHID–MgATP complex. The N-terminal AAA+ domain of ChlD is essential for complex formation, but some stability is preserved in the absence of the C-terminal integrin domain of ChlD, particularly if the intervening polyproline linker region is retained. Single molecule force spectroscopy (SMFS) was used to determine the factors that stabilize formation of the ChlID–MgADP complex at the single molecule level; ChlD was attached to an atomic force microscope (AFM) probe in two different orientations, and the ChlI subunits were tethered to a silica surface; the probability of subunits interacting more than doubled in the presence of MgADP, and we show that the N-terminal AAA+ domain of ChlD mediates this process, in agreement with the microscale thermophoresis data. Analysis of the unbinding data revealed a most probable interaction force of around 109 pN for formation of single ChlID–MgADP complexes. These experiments provide a quantitative basis for understanding the assembly and function of the Mg chelatase complex.

The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase

“…While ATPase activity has been observed and measured for the ChlI subunit [7–10], no ATPase activity has been observed for ChlD. ChlI hydrolyses MgATP 2− to provide the considerable power required to drive Mg 2+ insertion [5,11]. ChlD appears to act as an allosteric regulator in response to MgATP 2− concentrations [12], and the C-terminal domain of ChlD regulates the cooperative response to Mg 2+ concentrations in the Synechocystis sp.…”

Quantitative Evaluation of Viral Protein Binding to Phosphoinositide Receptors and Pharmacological Inhibition

“… 50 The interaction probability was also detected by classifying specific rupture events as possessing rupture lengths between 6 and 20 nm, whereas events with shorter or longer rupture lengths were classified as nonspecific interactions. 51 …”

Hexameric structure of the ATPase motor subunit of magnesium chelatase in chlorophyll biosynthesis

“…The BchD/ChlD subunit is composed of a C-terminal integrin I domain preceded by a proline-rich region and an N-terminal domain similar to BchI/ChlI, but possesses no ATPase activity. [18][19][20][21][22][23][24][25][26][27][28] Recently, it has been demonstrated that ChlD links the ATPase activity with the ChlH active site primarily through the integrin I domain. 29 Assembly of the BchD/ ChlD hexamer is generally assumed to be mediated by the N-terminal BchI/ChlI-homologous domain, while contribution from the integrin I domain is unclear.…”

“…However, due to limited resolution, it remains largely unknown how the hexamer is assembled. The BchD/ChlD subunit is composed of a C‐terminal integrin I domain preceded by a proline‐rich region and an N‐terminal domain similar to BchI/ChlI, but possesses no ATPase activity . Recently, it has been demonstrated that ChlD links the ATPase activity with the ChlH active site primarily through the integrin I domain .…”

“…The ChlI subunit from the cyanobacterium Synechocystis sp. PCC 6803 has been extensively characterized . The ChlI protomers can self‐assemble into a ring structure without ATP.…”