Structure of the mitochondrial creatine kinase octamer: high-resolution shadowing and image averaging of single molecules and formation of linear filaments under specific staining conditions.

Abstract

Abstract. The combination of high-resolution tantalum/tungsten (Ta/W) shadowing at very low specimen temperature (-250°C) under ultrahigh vacuum (<2 x 10 -9 mbar) with circular harmonic image averaging revealed details on the surface structure of mitochondrial creatine kinase (Mi-CK) molecules with a resolution <2.5 rim. Mi-CK octamers exhibit a cross-like surface depression dividing the square shaped projection of 10 × 10 nm into four equally sized subdomains, which correspond to the four dimers forming the octameric Mi-CK molecule. By a combination of positive staining (with uranyl acetate) and heavy metal shadowing, internal structures as well as the surface relief of Mi-CK were visualized at the same time at high resolution. Computational image analysis revealed only a single projection class of molecules, but the ability of Mi-CK to form linear filaments, as well as geometrical considerations concerning the formation of octamers by four equal, asymmetric dimers, suggest the existence of at least two distinct faces on the molecule. By image processing of Mi-CK filaments a side view of the octamer differing from the top-bottom projections of single molecules became evident showing a funnel-like access each form the top and bottom of the octamer connected by a central channel. The general structure of the Mi-CK octamer described here is relevant to the localization of the molecule at the inner-outer mitochondrial contact sites and to the function of Mi-CK as an "energy channeling" molecule.

The structure of mitochondrial creatine kinase and its membrane binding properties

“…Unidirectional shadowing led to the perspective impression of the surface of the octamer with its four hill-like quadrants. From the length of the molecules' shadow a quasi cubic three-dimensional shape of the octamer could be deduced [17]. The averaged image of rotary shadowed particles (Fig.…”

“…A broad spectrum of sample preparation techniques for electron microscopy was applied in combination with methods for image reconstruction to study the Mi-CK octamer [8,17]. Negatively stained octamers are squareshaped projections of about 10 nm in side-length with peripheral and central stain accumulation.…”

Functional Aspects of Creatine Kinase in Brain

“…Four major compart ments of CK are indicated: (I) strictly solu ble CK (CKc) freely equilibrating PC r/C r and ATP/A D P ratios in the cytosol; (2) cytosolic CK functionally coupled to gly colysis (CKS) [4,23,[25][26][27] on the produc ing side of the PCr circuit; (3) Mi-CK being functionally coupled to oxidative phospho rylation [l 1-13]; (4) 'cytosolic' CK, specifi cally associated with subcellular structures (CKa) at sites of high and fluctuating ATP requirements on the receiving end of the PCr circuit [for the different ATPases see 17,19,20,42]. Note that in resting muscle for example, the relative pool sizes of [ Also note that PCr and Cr are smaller and less charged molecules compared to the adenine nucleotides [for review see 4], At the mitochondrial side, a cube-like mitochondrial creatine kinase Mi-CK octamer molecule with an internal channel [8], shown to interact with inner (IM) as well as outer mitochondrial membranes (OM), thus stabilizing contacts between IM and OM in vitro [10], is depicted in con junction with the A TP/A D P translocator (ANT) of the IM, and with porin (P) of the OM, thus forming a multienzyme 'channel' [16] at the so-called 'mitochondrial energy transfer contact sites' [5]. The small black triangles (A) in the IM and in association with ANTs represent cardiolipin molecules.…”

“…Based on experiments showing that mito chondrial state 3 respiration can be effectively stimulated by creatine, leading to a net production of PCr by mito chondria [11][12][13], a functional coupling between Mi-CK and porin has also been postulated (see fig. 1) [5, for review see 4,6], Electron microscopy [8] and X-ray crys tallography [15] have shown that the cube-like Mi-CK octamer contains a central channel running parallel to the 4-fold axis through the entire molecule. This structure/function relationship, together with the results dis cussed above, led to a hypothesis that Mi-CK could act as a connecting module between ANT and porin at the mi tochondrial contact sites, thereby forming an efficient, tightly coupled multienzyme 'energy channel' that com bines the export of mitochondrial energy equivalents with the interconversion of matrixgenerated ATP to PCr [16] (see fig.…”

“…There, Mi-CK can directly convert the intramitochondrially produced ATP to PCr, which then is exported into the cytosol where it serves at relatively high concentration (5-40 mM, depending on the tissue) as an easily diffusable energy storage and transport metabolite [for review see 4, 6]. Mi-CK, which in contrast to the dimeric cytosolic CK isoenzymes forms highly symmetri cal, cube-like octameric structures [8,9], has the specific ability to peripherally bind to lipid membranes and, most importantly, to mediate contact site form ation between inner and outer mitochondrial membranes [10]. M ore over, Mi-CK is tightly functionally coupled to oxidative phosphorylation via the adenine nucleotide translocator (ANT) [11][12][13] which catalyses the antiport of ATP ver sus ADP through the inner membrane [14].…”